Multivolume devices, kits and related methods for quantification and detection of nucleic acids and other analytes

ABSTRACT

Provided are devices comprising multivolume analysis regions, the devices being capable of supporting amplification, detection, and other processes. Also provided are related methods of detecting or estimating the presence nucleic acids, viral levels, and other biological markers of interest.

CROSS-REFERENCE

This application is a divisional application of application Ser. No.13/467,482, filed May 9, 2012, which is a continuation-in-partapplication of application Ser. No. 13/440,371, filed on Apr. 5, 2012,which is a continuation-in-part application of application Ser. No.13/257,811, filed Sep. 20, 2011; which is the National Stage ofInternational Application No. PCT/US2010/028361, filed on Mar. 23, 2010,which claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication 61/262,375, filed on Nov. 18, 2009, and U.S. ProvisionalApplication No. 61/162,922, filed on Mar. 24, 2009, and U.S. ProvisionalApplication No. 61/340,872, filed on Mar. 22, 2010; application Ser. No.13/467,482 claims the benefit of U.S. Provisional Application No.61/518,601, filed May 9, 2011; application Ser. No. 13/440,371 claimsthe benefit of U.S. Provisional Application No. 61/516,628, filed Apr.5, 2011 and U.S. Provisional Application No. 61/518,601, filed May 9,2011; the content of all of which except application Ser. No. 13/467,482are hereby incorporated by reference in their entireties for any and allpurposes.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under grant numbersEB012946, GM074961, and OD003584 awarded by the National Institutes ofHealth and grant number CHE-0526693 awarded by the National ScienceFoundation. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 28, 2014, isnamed 45546-702.401_SL.txt and is 2,061 bytes in size.

TECHNICAL FIELD

The present application relates to the field of microfluidics and to thefields of detection and amplification of biological entities.

BACKGROUND

Real-time quantitative RT-PCR is an existing technique for monitoringviral load for HIV, HCV, and other viral infections. However, this testis cost-prohibitive in some resource-limited settings and can requiremultiple instruments, skilled technicians, and isolated rooms to preventcontamination. The test can thus be inaccessible to patients in someresource-limited settings. Moreover, the efficiency of RT-PCR, thequality of sample and selection of targets, and the methods forinterpretation of the data may in some cases present concerns for theaccuracy of quantifying RNA using RT-PCR.

Although dipstick-type devices may provide semiquantitative measurementsof viral load after amplification in resource limited settings, noquantitative test exists to resolve a 3-fold (i.e., appx. 0.5 log¹⁰)change in HIV RNA viral load, which change is considered clinicallysignificant. Accordingly, there is a long-felt need in the art fordevices and methods for quantitative measurement, estimates, and/or evendetection of viral load or other parameters.

SUMMARY

In meeting the described challenges, the present disclosure firstprovides devices. These devices comprise a first component comprising apopulation of first areas; a second component comprising a population ofsecond areas; the first and second components being engageable with oneanother such that relative motion between the first and secondcomponents exposes at least some of the first population of areas to atleast some of the second population of areas so as to form a pluralityof analysis regions. At least some of the analysis regions suitablydiffer in volume from others of the analysis regions.

Also disclosed are devices. The devices suitably include a firstcomponent comprising a population of first areas; a second componentcomprising a population of second areas; the first and second componentsbeing engageable with one another such that when the first and secondcomponents are in a first position relative to one another a fluidicpath is formed between at least some of the first areas and at leastsome of the second areas, and when the first and second components arein a second position relative to one another, the fluidic path isinterrupted so as to isolate at least some of the first areas from atleast some of the second areas.

Additionally provided are methods. These methods include distributingone or more target molecules from an original sample into a plurality ofanalysis regions, the distribution being effected such that at leastsome of the analysis regions are statistically estimated to each containa single target molecule, at least two of the analysis regions definingdifferent volumes; and effecting, in parallel, a reaction on at leastsome of the single target molecules.

Other methods presented in this disclosure include introducing an amountof a target molecule from an original sample into a device; effectingdistribution of the amount of the target molecule into at least twoisolated areas of the device, the at least two isolated areas definingvolumes that differ from one another; effecting a reaction on the targetmolecule so as to give rise to a reaction product in the at least twoisolated areas; and estimating, from the reaction product, the level ofa target in the original sample.

Also provided are methods, comprising distributing a plurality of targetmolecules—suitably nucleic acids—from an original sample into aplurality of analysis regions, the distribution being effected such thatat least some of the analysis regions are estimated to each contain asingle target molecule, at least two of the analysis regions definingdifferent volumes; and effecting, in parallel, a nucleic acidamplification reaction on at least some of the single target molecules.

Also disclosed are devices, comprising a first component comprising apopulation of first wells formed in a first surface of the firstcomponent, the population of wells being arranged in a radial pattern; asecond component comprising a population of second wells formed in afirst surface of the second component, the population of wells beingarranged in a radial pattern; the first and second components beingengageable with one another such that relative rotational motion betweenthe first and second components exposes at least some of the firstpopulation of wells to at least some of the second population of wellsso as to form a plurality of analysis regions, an analysis regioncomprising a first well and a second well in pairwise exposure with oneanother.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there are shown in the drawingsexemplary embodiments of the invention; however, the invention is notlimited to the specific methods, compositions, and devices disclosed. Inaddition, the drawings are not necessarily drawn to scale. In thedrawings:

FIG. 1 illustrates a rotationally-configured multivolume deviceaccording to the present disclosure;

FIG. 2 illustrates end-point fluorescence images of multivolume digitalRT-PCR performed on a rotational device according to the presentdisclosure;

FIG. 3 illustrates performance of digital RT-PCR with synthetic RNAtemplate on an exemplary multivolume device over a 4 log₁₀ dynamicrange;

FIG. 4 illustrates performance of an exemplary device;

FIG. 5 illustrates an exemplary device for multiplexed, multivolumedigital RT-PCR with high dynamic range;

FIG. 6 illustrates representative multivolume digital RT-PCR forquantification of HIV viral load in two patients' samples;

FIG. 7 illustrates a representative experiment performing RT-PCR of HIVviral RNA at an expected concentration of 51 molecules/mL in a RT-PCRmix;

FIG. 8 illustrates a representative negative control for HIV viral load;

FIG. 9 illustrates in tabular form detection and quantification data;

FIG. 10 presents a tabular summary of HIV quantification performance;

FIG. 11 presents a tabular summary of detection range data;

FIG. 12 shows an image, obtained with a iPhone 4S™ camera, of anexemplary multivolume device filled with LAMP reaction mix;

FIG. 13 shows a close-up of the center of the image in FIG. 12;

FIG. 14 presents a schematic view of a radially-arranged device forperforming MV digital PCR, the device design consisting of 160 wellseach at 125, 25, 5, and 1 nL. A sample is loaded from the center andafter filling the device components are relatively rotated so as toisolate wells—after reaction, wells containing template have enhancedsignal and are counted;

FIG. 15 presents, in tabular form, a summary of the specifications of anexemplary device according to the present disclosure;

FIG. 16 presents experimental results for MV digital PCR on an exemplarydevice using control DNA. Representative false color (shaded) images(lighter shading represents positive wells that showed at least a 3-foldincrease in intensity compared to negative wells) for solutions withinput concentrations of (a) 1500 molecules/mL and (b) 600,000molecules/mL (zoomed in on smaller wells). (c, d) Graphical summary ofall experiments comparing the input concentration, based on UV-vismeasurements (black curve), and observed concentrations using MV digitalPCR (x and +) over the entire dynamic range. Represented as (c) theactual concentration and (d) as a ratio to better show distribution ofresults. Stock samples were approximately 500, 1500, 8000, 20,000,30,000, 100,000, 600,000, and 3,000,000 molecules/mL. The confidenceintervals (CI) for the combined system (solid curves) indicate where 95%of the experiments should fall. CI curves for the individual volumes(dashed curves) are also provided to indicate over what range ofconcentration each volume contributes; and

FIG. 17 illustrates a separate analysis of 10 experimental results fordifferent well volumes with an input concentration of 30,000molecules/mL, showing distribution of measured concentrations for eachvolume and the overall agreement of results.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingfigures and examples, which form a part of this disclosure. It is to beunderstood that this invention is not limited to the specific devices,methods, applications, conditions or parameters described and/or shownherein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed invention. Also, as used in thespecification including the appended claims, the singular forms “a,”“an,” and “the” include the plural, and reference to a particularnumerical value includes at least that particular value, unless thecontext clearly dictates otherwise.

The term “plurality” as used herein, means more than one. When a rangeof values is expressed, another embodiment includes from the oneparticular value and/or to the other particular value. Similarly, whenvalues are expressed as approximations, by use of the antecedent about,it will be understood that the particular value forms anotherembodiment. All ranges are inclusive and combinable. All documents citedherein are incorporated herein by reference in their entireties for anyand all purposes.

In one embodiment, the present disclosure provides devices. Thesedevices suitably include a first component comprising a population offirst areas and a second component comprising a population of secondareas. The first and second components are suitably engageable with oneanother such that relative motion between the first and secondcomponents exposes at least some of the first population of areas to atleast some of the second population of areas so as to form a pluralityof analysis regions. As described herein, at least some of the analysisregions may suitably differ in volume from other analysis regions.

In some embodiments, the first and second components are engaged so asto permit rotational motion of one component relative to the othercomponent. This may be in the form of two plates that are rotatablyengaged with one another, as shown in exemplary FIG. 1. As shown inpanels A-D of that figure, the two plates may be rotatably engaged suchthat relative rotation between the plates gives rise to wells formed inthe plates aligning with one another (i.e., being placed into at leastpartial register) or de-aligning from one another.

Also as shown in that exemplary figure (FIG. 1, panel G), a device mayprovide analysis regions that differ from one another in volume. Forexample, the analysis regions (formed between FIG. 1 panels F and G byrelative rotational motion between two plates that gives rise topairwise exposure of wells formed in the plates to one another) shown inFIG. 1 have volumes of about 1, 5, 25, and 125 nL.

Although the analysis regions shown in FIG. 1 increase in volume withincreasing radial distance outward from axis of rotation between theplates, there is no requirement that analysis regions vary such a sizeand/or spatial manners. Other embodiments of the disclosed devicesfeature first and second components engaged so as to permit linearmovement of one component relative to the other component.

It should be understood that although exemplary FIG. 1 shows first andsecond components engaged with one another, the present disclosure isnot limited to devices that have only two components. For example, auser may construct a device that has first, second, and third componentsthat are engageable with one another. As but one example, the deviceshown in FIG. 1 may include a first well-bearing component that isengaged with one face of a second well-bearing component. The secondface of the second well-bearing component may, in turn, be engageablewith a third well-bearing component, such that the wells of the thirdcomponent may be placed into overlap with the wells of the second faceof the second well-bearing component. Such a structure may be in alayer-cake or sandwich form. These configurations enable increasedinformation density, as such configurations allow creation of additionalareas on a device where reactions and analysis may take place.

In an alternative embodiment, one surface of a component may be engagedwith two other components. For example, a base component having formedthereon first and second circular banks of wells that are separate fromone another may be engageable with [1] a first component that featureswells that may be placed into overlap with the first bank of wells ofthe base component and [2] a second component that features wells thatmay be placed into overlap with the second bank of wells of the basecomponent. In this way, a single device may feature multiple componentsso as to increase the number and diversity of reaction and/or analysislocations on the device.

The first and second components may be of a range of sizes. In someembodiments, at least one of the first or second components has athickness in the range of from about 10 micrometers to about 5000micrometers, or in the range of from about 50 micrometers to about 1000micrometers, or even from about 200 micrometers to about 500micrometers. These dimensions are particularly useful in applicationswhere a user may desire a reduced-size device or a device having arelatively compact form factor. Components may be formed of a glass, apolymer, and the like.

Soda-lime glass is considered especially suitable; other componentmaterials may also be used. Exemplary fabrication methods for suchdevices are set forth in Du et al., Lab Chip 2009, 9, 2286-2292. In someembodiments, components of the device are fabricated from a glasssubstrate by way of wet etching. In some other embodiments, the devicecan be fabricated from plastic materials, such as polycarbonate,Poly(methyl methacrylate) (PMMA), polypropylene, polyethylene, cycloolefin copolymer (COC), cyclo olefin polymer (COP), and fluorinatedpolymers including but not limited to fluorinated ethylene-propylene FEP(fluorinated ethylene-propylene), perfluoroakoxy (PFA) andpolytetrafluoroethylene (PTFE). Surfaces may be treated with methodssuch as silanization and physical deposition, such as vapor depositiontechniques. As one example, a surface of the device may be treated withdichlorodimethylsilane by vapor silanization. The surface of the devicecan be coated with silicone or fluorinated polymers. Reaction fluidsused in the devices may also include ingredients related to the surfacesof the devices. As one such example, bovine serum albumin is added to aPCR mixture to prevent adsorption and denaturation of molecules on asurface of the device.

In some embodiments (e.g., exemplary FIG. 5A), a component comprises aconduit that places at least some of the first population of wells intofluidic communication with the environment exterior to the component. Insome embodiments, a population of wells is characterized as beingradially disposed relative to a location on the first component, asshown in exemplary FIG. 5. Populations of wells may also be present in acircular pattern, a grid pattern, or virtually any other conformation.In some embodiments, a device (e.g., exemplary FIG. 5) may includeseveral populations of wells that are each in fluid communication withtheir own conduits, which in turn enables a user to introduce differentmaterials to different populations of wells. Exemplary FIG. 5 shows thisby reference to a device having five separate banks of wells, eachseparate bank of wells containing a different sample (samples I-V). Asshown in this exemplary figure, the banks of wells may be configuredsuch that a given bank of wells maintains a sample (e.g., sample I) inisolation from other samples (e.g., sample II). In the exemplary FIG. 5a, each bank of separate wells in in a form that is roughly a wedge or apie-slice in shape; banks of wells may have other layouts (grids, lines,and the like), depending on the device and on the user's needs. Thedevice may be configured such that each bank of wells has an inletconfigured to supply material (e.g., sample) to that bank of wells only.In this way, a device may have five banks of wells, each bank of wellsbeing supplied by one or more separate inlet. A bank of wells may, ofcourse, be supplied by one, two, three, or more inlets, depending on theuser's needs. A device may also be configured such that a given inletsupplies material to at least some wells in two or more separate banksof wells.

Certain embodiments of the disclosed devices feature components wherethe first areas are wells formed on (or in) a component. In someembodiments, a well suitably has a volume in the range of from about 0.1picoliter to about 10 microliters. In some variations, the second areasmay be wells. Such wells suitably have volumes in the range of fromabout 0.1 picoliter to about 10 microliters.

When areas are placed into exposure with one another so as to give riseto analysis regions, an analysis region may, in some embodiments, have avolume in the range of from about 0.1 picoliter to about 20 microliters.The volumes of two analysis regions may differ from one another. As oneexample, the ratio of the volumes defined by two analysis regions is inthe range of from about 1:1 to about 1:1,000,000, or from about 1:50 toabout 1:1,000, or from about 1:100 to about 1:500. In the example shownin FIG. 1, the ratio between some of the analysis region volumes is 1nl:125 nL=1:125.

The devices may also include an imager configured to capture at leastone image of an analysis region. The imager may be a camera, CCD, PMT,or other imaging device. Portable imagers, such as digital cameras andcameras on mobile devices such as smartphones/mobile phones areconsidered suitable imagers. The device may be configured such that theimager is positioned such that it may capture an image of some of theanalysis regions of a device or even an image of all of the analysisregions.

The devices may also be configured to display an image of an analysisregion for capture of at least one image by an imager. As one example,the device may be configured, as shown in exemplary FIG. 2, to presentor otherwise display the analysis regions in such a manner that theanalysis regions (including their contents) may be imaged. Exemplaryimages are shown in FIGS. 12 and 13, which images were obtained usingthe camera of an iPhone 4S™ mobile device.

The devices may also include a processor configured to estimate aconcentration of an analyte residing in one or more analysis regions.The processor may be present in the device itself; in such cases, thedevice may include the imager and a processor that is configured toestimate a concentration of an analyte residing in one or more analysisregions. This may be effected by, for example, a computer imagingroutine configured to take as an input an image of the analysis regionsof a device and then operate on that input (as described elsewhereherein) to estimate the concentration of an analyte in the one or moreanalysis regions. In one exemplary embodiment, the processor operates onan image of the analysis regions of the device and, from that, estimatesthe presence of an analyte in a sample.

As one example, a user may extract a blood sample from a subject todetermine whether a particular virus is present in the subject. The usermay then process the blood sample (e.g., cell isolation, cell lysis, andthe like) and assay the sample (e.g., via PCR) for the presence of aparticular nucleic acid that is a marker for the virus of interest. Theprocessor may then, by analyzing the image of the analysis regions inwhich the nucleic acid may be present, statistically estimate thepresence (if any) of the virus in the subject. The processor may,alternatively, be configured to detect the presence of the analyte in ayes/no fashion; this may be useful in situations where the user isinterested only in knowing whether the subject has a virus and is lessinterested in knowing the level of that virus in the subject. Furtherinformation regarding exemplary processing methods is found in Kreutz etal., JACS 2011 133: 17705-17712; Kreutz et al., Anal. Chem. 2011 83:8158-8168; and Shen et al., Anal. Chem. 2011 83: 3533-3540.

The disclosed devices are suitably configured to permit formation of 5,10, 100, 500 or more analysis regions. In some embodiments, the devicesare adapted to place at least about 10 first areas into pairwiseexposure with at least 10 second areas. This pairwise exposure in turneffects formation of 10 analysis regions. The devices may also beadapted so as to be capable of placing at least about 100 first areasinto pairwise exposure with at least 100 second areas, or even placingat least about 200 first areas into pairwise exposure with at least 200second areas. Exemplary FIG. 1 shows (by way of the “slip” rotationalmotion shown between FIG. 1 panels C and D) placement of filled wells(darkened circles) into exposure with unfilled wells (unfilled circles).

Devices according to the present disclosure may also include a quantityof a reagent disposed within the device. The reagent may be a salt, abuffer, an enzyme, and the like. Reagents that are useful in anamplification reaction are considered especially suitable. Such reagentsmay be disposed within an area of the device in dried or liquid form.Reagents may also be disposed within the device within a well; fluidreagents may be preloaded into wells where the reagents remain until thedevice is used.

In one such embodiment, a sample is loaded into a first population ofwells on a first component of a device. The device may also include asecond population of wells on a second component of the device, thesecond population of wells being pre-filled with a reagent selected toreact with the sample. Relative motion between the first and secondcomponents exposes at least some of the first population of wells to atleast some of the second population of wells (e.g., FIG. 1), and thepre-stored reagent may then react with the sample in individual analysisregions formed from first and second wells exposed to one another.

The present disclosure provides other devices. These devices suitablyinclude a first component comprising a population of first areas and asecond component comprising a population of second areas, with the firstand second components suitably being engageable with one another suchthat when the first and second components are in a first positionrelative to one another a fluidic path is formed between at least someof the first areas and at least some of the second areas. The devicesare also suitably configured such that when the first and secondcomponents are in a second position relative to one another, the fluidicpath is interrupted so as to isolate at least some of the first areasfrom at least some of the second areas.

One such exemplary embodiment is shown by FIG. 1. As shown in thatfigure, first and second components (well-bearing plates, in thisfigure) are engaged with one another (right side of panel A). In a firstposition (panels B and C), wells formed in the upper component (shownwith dotted lines) and wells formed in the lower component (shown bysolid lines) form a fluidic path, which path is shown by the fluidfilling illustrated in panel C. The filling may be effected by an inlet(not shown in FIG. 1) that is formed in (or through) a component so asto connect a well of the component to the exterior of the device.

In a second position (shown in panel D), the fluidic path is interruptedby way of relative motion of the well-bearing components so as toisolate some of the wells that formerly defined the fluidic path fromone another. In this way, the device allows a user to [1] introduce amaterial into multiple areas (e.g., wells) and then [2] isolate thoseareas from one another so as to allow processing of that material inindividualized quantities. Suitable components and the characteristicsof these components (e.g., wells, well volumes) are described elsewhereherein. It should be understood that the devices may include embodimentswhere two or more first areas may differ from one another in terms ofvolume. For example, a first component may include wells of 1 nL, 10 nL,and 100 nL formed therein. Likewise, the devices may include embodimentswhere two or more second areas may differ from one another in terms ofvolume. For example, a second component may include wells of 1 nL, 10nL, and 100 nL formed therein. As shown by exemplary panel C of FIG. 1,the fluidic path may comprise at least one first area at least partiallyexposed to (e.g., overlapping with) at least one second area. Theoverlap between first and second areas may give rise to analysisregions, which analysis regions may (as described elsewhere herein) havedifferent volumes from one another.

In some particularly suitable embodiments, the fluidic path isconfigured to permit the passage of aqueous media. This may beaccomplished, for example, by placing a layer of material (e.g.,lubricating fluid or oil) between the first and second components. Asexplained in the other documents cited herein, the layer of lubricatingoil may act to isolate a well formed in the first component from otherwells formed in the first component and also from wells formed in thesecond component, except when those wells are exposed (e.g., placed intoat least partial register) to one another. The lubricating oil may bechosen such that it does not permit the passage of aqueous media. Insome embodiments, the lubricating fluid may be mineral oil, tetradecane,long chain hydrocarbon, silicone oil, fluorocarbon, and the like, aswell as combinations of the foregoing.

It should be understood that in some embodiments, an oil or othernon-aqueous material may also be disposed within a well. This may beshown by reference to exemplary FIG. 1. In one embodiment, certain firstand second wells may be filled with an aqueous material (panel C in FIG.1). Other wells on the device that are not filled with the aqueousmaterial may be filled with an oil (not shown). When the fluidic pathbetween the aqueous-filled wells is broken (panels C and D), theaqueous-filled wells are exposed pairwise to wells that are filled withoil.

In some of devices, an analysis region may include an isolated firstarea or an isolated second area. In one such embodiment, the firstcomponent comprises wells formed therein and the second compartmentcomprises wells formed therein. The components may be positioned suchthat (e.g., FIG. 1, panel C) at least some of the first and second wellsform a fluidic path. The components may then be positioned such that thefluidic path is interrupted and, further, that some of the first wellsare (not shown) positioned opposite to a flat (i.e., non-well bearing)portion of the second component, and some of the second wells arepositioned opposite to a flat (i.e., non-well bearing) portion of thefirst component. In other embodiments, an analysis region may comprisean isolated first area and an isolated second area that are exposed onlyto one another. This is shown by panel D of non-limiting FIG. 1.

Also provided are methods. These methods suitably include distributingone or more target molecules from an original sample into a plurality ofanalysis regions, the distribution being effected such that at leastsome of the analysis regions are statistically estimated to each containa single target molecule. Embodiments where at least two of the analysisregions have different volumes from another are considered especiallysuitable. In some embodiments, the methods include effecting a reactionon at least some of the single target molecules. The reaction may takeplace in parallel, i.e., the reaction occurs on two or more targetmolecules at the same time. The reaction may be also performed inmultiple analysis regions at the same time. It should be understood thatdifferent types reactions (e.g., amplification, lysing) may take placeat the same time at different analysis regions.

The distribution may be effected by dividing an area within which one ormore target molecules resides into at least two analysis regions. PanelsC and D of FIG. 1 are illustrative of this aspect of the method. Inpanel C, a fluid containing one or more target molecules is introducedinto a fluidic path that comprises, as described elsewhere herein, wellsformed in first and second components. When the fluidic path isinterrupted (panel D of FIG. 1), the fluid is subdivided into variousanalysis regions. The volumes of the analysis regions and thetarget-molecule containing fluid itself may be configured such that atleast some of the analysis regions each contain (or at estimated to eachcontain) a single target molecule.

The user may also estimate the concentration of a target compound in theoriginal sample. This estimation may be effected by application of mostprobable number theory, as described in Shen et al., JACS 2011 133:17705-17712, Kreutz et al., Analytical Chemistry 2011 83: 8158-8168, andShen et al., “Digital Isothermal Quantification of Nucleic Acids viaSimultaneous Chemical Initiation of Recombinase Polymerase AmplificationReactions on SlipChip”, Analytical Chemistry 2011 83:3533-3540. Theestimation may be performed such that the estimation has a lowerdetection limit, at a 95% confidence value, of more than about 0.1molecules/mL, and an upper level of quantification of less than about10¹² molecules/mL.

In some embodiments, the target molecule comprises a target nucleicacid, and the method is capable of estimating the concentration of thetarget nucleic acid in the original sample with at least about 3-foldresolution for original samples with concentrations of about 500molecules or more of target nucleic acid per milliliter.

Nucleic acids and proteins are considered especially suitable targetmolecules. In some embodiments, the reaction is an amplification, suchas a nucleic acid amplification. The amplification may be performedwithin 5, 10, 20, 50, 100, 500, or even 1000 analysis regions. Theamplification may be performed in such a way that amplification occursin at least two analysis regions at the same time, although it is notnecessary that amplification begin or end at the same time in thedifferent analysis regions. The amplification may be performed in anessentially isothermal manner such that the process takes place within atemperature range of plus or minus about 10 degrees C. For example, theamplification may take place at within 10 degrees C. of ambientconditions.

A variety of amplification techniques may be used, as describedelsewhere herein. Some such suitably techniques include a polymerasechain reaction, a room-temperature polymerase chain reaction, a nestedpolymerase chain reaction, a multiplex polymerase chain reaction, anarbitrarily primed polymerase chain reaction, a nucleic acidsequence-based amplification, a transcription mediated amplification, astrand displacement amplification, a branched DNA probe targetamplification, a ligase chain reaction, a cleavase invaderamplification, an anti DNA-RNA hybrid antibody amplification, and thelike.

An analysis region may, as described elsewhere herein, comprise firstand second areas in pairwise exposure with one another. The user mayeffect relative motion between a first component comprising a pluralityof first areas and a second component comprising a plurality of secondareas, the relative motion placing at least one first area and at leastone second area into pairwise exposure with one another to define atleast one analysis region. This is illustrated in FIG. 1, panels C andD, where first and second areas are placed into pairwise exposure withone another so as to define analysis regions. The relative motion mayplace at least about 10 first areas into pairwise exposure with at leastabout 10 second areas, or may even place at least about 100 first areasinto pairwise exposure with at least about 100 second areas.

The instant disclosure also provides methods. These methods includeintroducing an amount of a target molecule from an original sample intoa device; effecting distribution of the amount of the target moleculeinto at least two isolated areas of the device, the at least twoisolated areas defining volumes that differ from one another; effectinga reaction on the target molecule so as to give rise to a reactionproduct in the at least two isolated areas; and estimating, from thereaction product, the level of target molecule in the original sample.

The target molecule may be, for example, a nucleic acid. The methods mayalso include contacting an amplification reagent—such as a reagentuseful in PCR—with the nucleic acid. In some embodiments, at least oneisolated area is estimated to contain one nucleic acid molecule, asdescribed elsewhere herein. One particularly suitable reaction toperform within the disclosed methods is nucleic acid amplification;suitable amplification techniques are described elsewhere herein. Thenucleic acid amplification may be essentially isothermal, as describedelsewhere herein.

The methods may also include estimating the level of a nucleic acid inthe original sample. In some embodiments, at least one of the isolatedareas is estimated to comprise about one molecule of nucleic acid. Thisfacilitates application of the estimation methods described in Kreutz etal., JACS 2011 133: 17705-17712; Kreutz et al., Anal. Chem. 2011 83:8158-8168; and Shen et al., Anal. Chem. 2011 83: 3533-3540. Thedisclosed methods may be capable of estimating the concentration oftarget nucleic acid in the original sample with at least about 3-foldresolution for original samples with concentrations of about 500molecules or more of target nucleic acid per milliliter.

An isolated area, e.g., a well, may suitably have a volume in the rangeof from about 1 picoliter to about 10 microliters, as describedelsewhere herein. Volumes in the range of from about 1 nL to about 500nL, or even from about 5 nL to about 100 nL are considered suitable.

Distribution of some amount of target molecules may be effected byeffecting relative motion between a first and second component so as todistribute the amount of the target molecule into at least two isolatedareas, as described elsewhere herein and as shown by exemplary FIG. 1panels B-D. The relative motion may give rise to the amount of thetarget molecule being divided among at least 10 isolated areas, or evenamong at least 50 isolated areas.

According to the disclosed methods, a reaction may be effected at two ormore areas essentially simultaneously. The reactions need notnecessarily (but can be) the same in two or more areas. For example, auser may effect an amplification reaction at three areas while effectinga different reaction (e.g., denaturing) at three other areas.

Other disclosed methods include distributing a plurality of targetmolecules from an original sample into a plurality of analysis regions,the distribution being effected such that at least some of the analysisregions are estimated to each contain a single target molecule, and atleast two of the analysis regions defining different volumes; effecting,in parallel, a nucleic acid amplification reaction on at least some ofthe single target molecules. Suitable amplification techniques aredescribed elsewhere herein. The amplification may, in some embodiments,be effected essentially isothermally.

In some embodiments, the methods further include removing a product ofthe nucleic acid amplification reaction. Such recovery may be carriedout, in some embodiments, by accessing individual wells of a device. Insome embodiments, recovery is achieved by combining material frommultiple wells, for example by placing a device into the loadingposition and using a carrier fluid (including a gas) to expel thematerial from the device. Recovery may also be accomplished by pipettingmaterial out of a device. Such recovery may be used for additionalanalysis of nucleic acid products, such as sequencing, genotyping,analysis of methylation patterns, and identification of epigeneticmarkers.

Recovered material may be removed from the device. In some embodiments,recovered material may be transferred to another device, or anotherregion of the same device. Amplification may be carried out by themethods described herein or by other methods known in the art or bytheir combinations. As one non-limiting example, a user may detect thepresence of a target nucleic acid, e.g., by PCR. Once the presence ofthe target is confirmed, the user may remove the product from thedevice. This may be accomplished by pipetting the product out of anindividual well and transferring that product to another device orcontainer. The recovered product may be further processed, e.g.,sequenced. A variety of sequencing methods are known to those ofordinary skill in the art, including Maxam-Gilbert sequencing,chain-termination sequencing, polony sequencing, 454 Sequencing™, SOLiDSequencing™, ion semiconductor sequencing, nanoball sequencing,Helioscope™ sequencing, nanopore sequencing, single-molecule SMRT™sequencing, single molecule real time sequencing (RNAP), and the like.

The present disclosure also provides devices. These devices include afirst component comprising a population of first wells formed in a firstsurface of the first component, the population of wells being arrangedin a radial pattern; a second component comprising a population ofsecond wells formed in a first surface of the second component, theplurality of wells being arranged in a radial pattern; the first andsecond components being engageable with one another such that relativerotational motion between the first and second components exposes atleast some of the first population of wells to at least some of thesecond population of wells so as to form a plurality of analysisregions, an analysis region comprising a first well and a second well inpairwise exposure with one another.

In some embodiments, at least two analysis regions have volumes thatdiffer from one another, as described elsewhere herein. The firstcomponent may include a channel having an inlet, the channel configuredso as to place at least some of the first wells into fluid communicationwith the environment exterior to the channel. The inlet may reside in asurface of the first component other than the surface of the firstcomponent in which the first wells are formed. In this way, when the twocomponents are assembled together such that the wells of the firstcomponent face the wells of the second component, the user may fill thewells of the first component without dissembling the device.

It should be understood that the second component may also include achannel and inlet, configured such that the channel inlet is formed in asurface of the second component other than the surface of that componentin which the wells reside.

The devices may include, e.g., from about 10 to about 10,000 firstwells. The devices may also include, e.g., from about 10 to about 10,000second wells.

The disclosed methods may further include estimating the level,presence, or both of the one or more nucleic acids in the biologicalsample. Estimating may comprise (a) estimating the presence or absenceof the one or more nucleic acids in two or more wells of differentvolumes and (b) correlating the estimated presence or absence of the oneor more nucleic acids in the two or more wells of different volumes to alevel of the one or more nucleic acids in the biological sample, or,alternatively, to the level of some other target in a biological sampleor even in a subject. Exemplary estimation methods are describedelsewhere herein.

The present disclosure provides estimating the level of a target presentin a sample (e.g., estimating viral load in a subject by determining thepresence or concentration of a nucleic acid marker in a sample). Thepresent disclosure, however, also provides detecting the presence of atarget in a sample so as to provide the user with a yes/no determinationconcerning whether a particular analyte is present in a subject. Inthese embodiments, the user may perform a reaction (e.g., amplification,labeling) on a sample and merely assay for the presence of a “positive”result of the reaction.

One estimation method is provided in Kreutz et al., Anal. Chem. 2011 83:8158-8168. As explained in that publication, theoretical methods may beused—in conjunction with software analysis tools—to design and analyzemultivolume analysis devices. Multivolume digital PCR (“MV digital PCR”)is a reaction that is especially amenable to these methods. MV digitalPCR minimizes the total number of wells required for “digital” (singlemolecule) measurements while also maintaining high dynamic range andhigh resolution.

As one illustrative example, a multivolume device having fewer than 200total wells is predicted to provide dynamic range with a 5-foldresolution. Without being bound to any particular theory, thisresolution is similar to that of single-volume designs that useapproximately 12,000 wells.

Mathematical techniques, such as application of the Poisson distributionand binomial statistics, may be used to process information obtainedfrom an experiment and to quantify performance of devices. Thesetechniques were experimentally validated using the disclosed devices.

MV digital PCR has been demonstrated to perform reliably, and resultsfrom wells of different volumes agreed with one another. In using thedevices, no artifacts due to different surface-to-volume ratios wereobserved, and single molecule amplification in volumes ranging from 1 to125 nL was self-consistent.

An exemplary device according to the present disclosure was constructedto meet the testing requirements for measuring clinically relevantlevels of HIV viral load at the point-of-care (in plasma, <500molecules/mL to >1,000,000 molecules/mL). The predicted resolution anddynamic range was experimentally validated using a control sequence ofDNA, as described in Kreutz et al., Anal. Chem. 2011 83: 8158-8168.

The estimation theory applied in the above publication may be summarizedas follows. First, there are two assumptions that are maintained: (1)having at least one target molecule in a well is necessary andsufficient for a positive signal, and (2) target molecules do notinteract with one another or device surfaces, to avoid biasing theirdistribution. At the simplest level of analysis, when molecules are atlow enough densities that there is either 0 or 1 molecule within a well,concentrations can be estimated simply by counting wells displaying a“positive” signal. Under the above assumptions, Poisson and binomialstatistics may be used to obtain quantitative results from experimentsresulting in one positive well to experiments resulting in one negativewell. The Poisson distribution (eq. 1), in the context of digital PCR,gives the probability, p, that there are k target molecules in a givenwell based on an average concentration per well, v·λ, where v is thewell volume (mL) and λ is the bulk concentration (molecules/mL). Indigital PCR, the same readout occurs for all k>0, so if k=0, then eq. 1simplifies to give the probability, p, that a given well will notcontain target molecules (the well is “negative”).p=((v·λ)^(k) ′e ^(−(v·λ)))/k!,And for k=0 (empty well), p=e ^(−(v·λ))  (1)

In single-volume systems, the number of negative wells, b, out of totalwells, n, can serve as an estimate for p, so expected results can beestimated from known concentrations, or observed results can be used tocalculate expected concentrations (eq. 2).b=n·e ^(−(v·λ)) or λ=−ln(b/n)/v  (2)

The binomial equation is used to determine the probability, P, that aspecific experimental result (with a specific number of negatives, b,and positives, n−b, out of the total number of wells, n, at each volume)will be observed, on the basis of λ (eq. 3),

$\begin{matrix}{{{{where}\mspace{14mu}\begin{pmatrix}n \\b\end{pmatrix}} = \frac{n!}{{b!}{\left( {n - b} \right)!}}}{P = {{\begin{pmatrix}n \\b\end{pmatrix} \cdot p^{b} \cdot \left( {1 - p} \right)^{n - b}}\mspace{14mu}{or}}}{P = {\begin{pmatrix}n \\b\end{pmatrix} \cdot \left( e^{{- v} \cdot \lambda} \right)^{b} \cdot \left( {1 - e^{{- v} \cdot \lambda}} \right)^{n - b}}}} & (3)\end{matrix}$

An incomplete analysis of multivolume systems may be performed by simplyselecting a single volume and analyzing it as described above; this isthe approach that has typically been taken in serial dilution systems.The single volume that minimizes the standard error is generally chosen;this typically occurs when 10-40% of wells are negative. This method,however, does not utilize the information from the other “dilutions” (orvolumes), and would require using different dilutions for differentsample concentrations. Combining the results from wells of differentvolumes fully minimizes the standard error and provides high-qualityanalysis across a very large dynamic range. This is achieved by properlycombining the results of multiple binomial distributions (one for eachvolume); specifically, the probability of a specific experimental resultP (defined above) is the product of the binomials for each volume (eq.4), where P is defined as a function of the bulk concentration λ,P=f(λ),

$\begin{matrix}{{f(\lambda)} = {P = {\prod{\begin{pmatrix}n_{i} \\b_{i}\end{pmatrix} \cdot \left( e^{{- v_{i}} \cdot \lambda} \right)^{b_{i}} \cdot \left( {1 - e^{{- v_{i}} \cdot \lambda}} \right) \cdot \left( {1 - e^{{- v_{i}} \cdot \lambda}} \right)^{n_{i} - b_{i}}}}}} & (4)\end{matrix}$

For a given set of results, the MPN is found by solving for the value ofλ that maximizes P. In general, taking the derivative of an equation andsolving for zero provides the maximum and/or minimum values of thatequation; as a binomial distribution (and subsequently the product ofbinomials) has only a single maximum, solving the derivative of eq. 4for zero provides the “most probable” concentration. The standarddeviation, σ, is more appropriately applied to ln(λ) than to λ, becausethe distribution of P based on ln(λ) is more symmetrical than that forλ. In addition, this approach provides better accuracy for lowconcentrations by enforcing the constraint that concentrations must bepositive. Thus, a change of variables is needed during the derivationsso σ can be found for ln(λ). Therefore, f(λ) (eq. 4) is converted toF(∧) (eq. 5), where θ=e^(−v) and ∧=ln(λ).

$\begin{matrix}{{F(\Lambda)} = {P{\prod{\begin{pmatrix}n_{i} \\b_{i}\end{pmatrix} \cdot \left( \theta_{i}^{e^{\Lambda}} \right)^{b_{i}} \cdot \left( {1 - \theta_{i}^{e^{\Lambda}}} \right)^{n_{i} - b_{i}}}}}} & (5)\end{matrix}$

The derivative is easier to handle if the natural log is applied to eq.5, as the individual components are separated, but the overall result isunchanged (eq. 6).

$\begin{matrix}{{L(\Lambda)} = {{\ln\;{F(\Lambda)}} = {\sum\limits_{i = 1}^{m}\left( {{\ln\begin{pmatrix}n_{i} \\b_{i}\end{pmatrix}} + {b_{i} \cdot e^{\Lambda} \cdot {\ln\left( \theta_{i} \right)}} + {\left( {n_{i} - b_{i}} \right) \cdot {\ln\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}}} \right)}}} & (6)\end{matrix}$

The first derivative is then

$\frac{\partial{L(\Lambda)}}{\partial\Lambda} = {\sum\limits_{i = 1}^{m}\left( {0 + {b_{i} \cdot e^{\Lambda} \cdot {\ln\left( \theta_{i} \right)}} - {\frac{\left( {n_{i} - b_{i}} \right) \cdot e^{\Lambda} \cdot \theta_{i}^{e^{\Lambda}}}{1 - \theta_{i}^{e^{\Lambda}}} \cdot {\ln\left( \theta_{i} \right)}}} \right)}$

ln(θi) can be replaced with vi:

$= {e^{\Lambda} \cdot {\sum\limits_{i = 1}^{m}\left( {{{- b_{i}} \cdot v_{i}} + \frac{\left( {n_{i} - b_{i}} \right) \cdot v_{i} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}} \right)}}$

substituting (n_(i)−t_(i)) for b_(i) (where t_(i) is the number ofpositive wells):

$= {e^{\Lambda} \cdot {\sum\limits_{i = 1}^{m}\left( {{{- n_{i}} \cdot v_{i}} + {t_{i} \cdot v_{i}} + \frac{t_{i} \cdot v_{i} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}} \right)}}$

rearranging to put all t_(i)'s over the denominator

$= {e^{\Lambda} \cdot {\sum\limits_{i = 1}^{m}\left( {{{- n_{i}} \cdot v_{i}} + \frac{t_{i} \cdot v_{i}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)} - \frac{t_{i} \cdot v_{i} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)} + \frac{t_{i} \cdot v_{i} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}} \right)}}$

and simplifying and rearranging in terms of b_(i)

$\begin{matrix}{\frac{\partial{L(\Lambda)}}{\partial\Lambda} = {e^{\Lambda} \cdot {\sum\limits_{i = 1}^{m}\left( {{{- n_{i}} \cdot v_{i}} + \frac{\left( {n_{i} - b_{i}} \right) \cdot v_{i}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}} \right)}}} & (7)\end{matrix}$

Setting eq. 7 equal to 0, re-substituting λ, and rearranging then giveseq. 8. By solving eq. 8 for λ, the expected concentration can bedetermined from the number of empty wells. This can be done using anysolver function; the code MVdPCR_MLE.m (described in Kreutz et al.,Analytical Chemistry 2011 83: 8158-8168) performs this step using aglobalized Newton method.

$\begin{matrix}{{\sum\limits_{i = 1}^{m}{n_{i} \cdot v_{i}}} = {\sum\limits_{i = 1}^{m}\frac{\left( {n_{i} - b_{i}} \right) \cdot v_{i}}{\left( {1 - e^{{- v_{i}} \cdot \lambda}} \right)}}} & (8)\end{matrix}$

The standard error, σ, for a result can be found using the Fisherinformation, I(X), for ln(λ), 44 requiring the change of variable to ∧.The Fisher information is defined in eq. 9, where E[ ] represents theexpected value of the given variable.

$\begin{matrix}\begin{matrix}{\frac{1}{variance} = {\frac{1}{\sigma^{2}} = {{I(\Lambda)} = {- {\int{\frac{\partial^{2}{I(\Lambda)}}{\partial\Lambda^{2}}{f\left( {x;\theta} \right)}d\; x}}}}}} \\{= {E\left\lbrack {- \frac{\partial^{2}{L(\Lambda)}}{\partial\Lambda^{2}}} \right\rbrack}}\end{matrix} & (9)\end{matrix}$

In eq. 10, the second derivative of eq. 6 is found.

$\begin{matrix}\begin{matrix}{\frac{\partial^{2}{L(\Lambda)}}{\partial\Lambda^{2}} = {{e^{\Lambda} \cdot {\sum\limits_{i = 1}^{m}\left( {{{- n_{i}} \cdot v_{i}} + \frac{\left( {n_{i} - b_{i}} \right) \cdot v_{i}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}} \right)}} +}} \\{e^{\Lambda} \cdot {\sum\limits_{i = 1}^{m}\left( \frac{e^{\Lambda} \cdot \left( {n_{i} - b_{i}} \right) \cdot \theta_{i}^{e^{\Lambda}} \cdot v_{i} \cdot \left( {\ln\;\theta} \right)}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)^{2}} \right)}} \\{= {{e^{\Lambda} \cdot {\sum\limits_{i = 1}^{m}\left( {{n_{i} \cdot v_{i}} - \frac{\left( {n_{i} - b_{i}} \right) \cdot v_{i}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}} \right)}} - {\left( e^{\Lambda} \right)^{2} \cdot {\sum\limits_{i = 1}^{m}\left( \frac{\left( {n_{i} - b_{i}} \right) \cdot v_{i}^{2} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)^{2}} \right)}}}}\end{matrix} & (10)\end{matrix}$

Using this expression in eq. 9 to then find the inverse variance giveseq. 11

$\begin{matrix}{\begin{matrix}{\frac{1}{\sigma^{2}} = {- {E\left\lbrack {{e^{\Lambda} \cdot {\sum\limits_{i = 1}^{m}\left( {{n_{i} \cdot v_{i}} - \frac{\left( {n_{i} - b_{i}} \right) \cdot v_{i}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}} \right)}} -} \right.}}} \\\left. {\left( e^{\Lambda} \right)^{2} \cdot {\sum\limits_{i = 1}^{m}\left( \frac{\left( {n_{i} - b_{i}} \right) \cdot v_{i}^{2} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)^{2}} \right)}} \right\rbrack \\{= {{{- e^{\Lambda}} \cdot {\sum\limits_{i = 1}^{m}\left( {{n_{i} \cdot v_{i}} - \frac{\left( {n_{i} - {E\left\lbrack b_{i} \right\rbrack}} \right) \cdot v_{i}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}} \right)}} +}} \\{\left( e^{\Lambda} \right)^{2} \cdot {\sum\limits_{i = 1}^{m}\left( \frac{\left( {n_{i} - {E\left\lbrack b_{1} \right\rbrack}} \right) \cdot v_{i}^{2} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)^{2}} \right)}}\end{matrix}{{With}\mspace{14mu}{E\left\lbrack b_{i} \right\rbrack}\mspace{14mu}{coming}\mspace{14mu}{from}\mspace{14mu}{eq}\mspace{14mu} 2}\begin{matrix}{\;{= {{{- e^{\Lambda}} \cdot {\sum\limits_{i = 1}^{m}\left( {{n_{i} \cdot v_{i}} - \frac{\left( {n_{i} - {n_{i} \cdot \theta_{i}^{e^{\Lambda}}}} \right) \cdot v_{i}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}} \right)}} +}}} \\{\left( e^{\Lambda} \right)^{2} \cdot {\sum\limits_{i = 1}^{m}\left( \frac{\left( {n_{i} - {n_{i} \cdot \theta_{i}^{e^{\Lambda}}}} \right) \cdot v_{i}^{2} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)^{2}} \right)}} \\{= {{{- e^{\Lambda}} \cdot {\sum\limits_{i = 1}^{m}\left( {{n_{i} \cdot v_{i}} - {n_{i} \cdot v_{i} \cdot \frac{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)}}} \right)}} +}} \\{\left( e^{\Lambda} \right)^{2} \cdot {\sum\limits_{i = 1}^{m}\left( \frac{n_{i} \cdot \left( {1 - \theta_{i}^{e^{\Lambda}}} \right) \cdot v_{i}^{2} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)^{2}} \right)}} \\{= {{\left( e^{\Lambda} \right)^{2} \cdot {\sum\limits_{i = 1}^{m}\left( \frac{n_{i} \cdot v_{i}^{2} \cdot \theta_{i}^{e^{\Lambda}}}{\left( {1 - \theta_{i}^{e^{\Lambda}}} \right)} \right)}} = {\lambda^{2} \cdot {\sum\limits_{i = 1}^{m}\left( \frac{n_{i} \cdot v_{i}^{2} \cdot e^{{- v_{i}}\; \cdot \lambda}}{\left( {1 - e^{{- v_{i}} \cdot \lambda}} \right)} \right)}}}} \\{= {\lambda^{2} \cdot {\sum\limits_{i = 1}^{m}\left( \frac{n_{i} \cdot v_{i}^{2}}{\left( {e^{v_{i} \cdot \lambda} - 1} \right)} \right)}}}\end{matrix}} & (11)\end{matrix}$

This ultimately gives the standard error (eq. 12), from which confidenceintervals can be generated (eq. 13), where Z is the upper critical valuefor the standard normal distribution.

$\begin{matrix}{\sigma = \frac{1}{\sqrt{\lambda^{2} \cdot {\sum\frac{v_{i}^{2} \cdot n_{i}}{e^{v_{i} \cdot \lambda} - 1}}}}} & (12) \\{{CI} = {{\ln(\lambda)} \pm {Z \cdot \sigma}}} & (13)\end{matrix}$

One aspect of the disclosed devices achieves a certain resolution (thatis, to distinguish a certain difference in concentration) at certainconcentrations. As mentioned above for HIV viral load monitoring, asystem suitably achieves a 3-fold resolution for as low as 500molecules/mL. To correctly resolve two different concentrations, thepotential for false positives (Type I error) and false negatives (TypeII error) may be considered. Samples suitably give results at thedesired confidence level (1-α, measure of Type I error) and demonstratethis confidence level again and again (Power: 1-β, measure of Type IIerror).

When comparing two results, the null hypothesis is that the results comefrom samples that have statistically the same concentration. α is theprobability that two results that are determined to be statisticallydifferent are in fact from the same sample, thus resulting in a falsepositive. A 95% confidence level would correspond to α=0.05 and anaccepted false positive rate of 5%. The power level measures theprobability, β, that samples that are statistically different at thedesired confidence level give results that fall below this confidencelevel. A 95% power level would correspond to β=0.05 and thus an acceptedfalse negative rate of 5%. For the exemplary analysis described herein,the 3-fold resolution is defined such that samples with a 3-folddifference in concentration (e.g., 500 and 1500 molecules/mL) givesresults that are statistically different with at least 95% confidence(α<0.05, less than 5% false positives) at least 95% of the time (powerlevel of 95%, β<0.05, no more than 5% false negatives). The Z-test (eq.14) was chosen to measure the confidence level, where λ and σ arecalculated using eqs. 8 and 12, respectively, for a set of two results(the specific number of negatives, b_(i), out of the total number ofwells, n_(i), at each volume i of wells). The Z-test measures theprobability that results are statistically different, by assuming thatthe test statistics (left side of eq. 14) can be approximated by astandard normal distribution, so Z corresponds to a known probability.Power level is measured by simulating results from two different samplesand determining the probability that they will give results that atleast meet the desired confidence level.

$\begin{matrix}{{\frac{\lambda_{1} - \lambda_{2}}{\sqrt{\sigma_{1}^{2} + \sigma_{2}^{2}}} = Z},\mspace{14mu}{{{for}\mspace{14mu} 95\%\mspace{14mu}{confidence}\frac{\lambda_{1} - \lambda_{2}}{\sqrt{\sigma_{1}^{2} + \sigma_{2}^{2}}}} > 1.96}} & (14)\end{matrix}$

A multivolume device was designed with 160 wells each at volumes of 125,25, 5, and 1 nL (FIG. 15). A radial layout of wells (FIG. 14) providesan efficient use of space when wells of significantly different volumesare used. In the initial orientation of the radial multivolume device,the wells are aligned to create a continuous fluidic path that allowsall of the sample wells to be filled in one step using dead end filling.The components of the device can then be rotationally slipped ortranslated (by ˜8° to simultaneously isolate each well and also overlapthe well with an optional corresponding thermal expansion well (FIG.14). This device has a LDL of 120 molecules/mL and a dynamic range whereat least 3-fold resolution is achieved from 520 to 3,980,000molecules/mL (FIG. 15). A control 631 bp sequence of DNA was used tovalidate the MV digital PCR approach. The initial concentration of thisstock solution was determined by UV-vis, and the stock was then dilutedto levels required for testing of the chip. Concentrations were testedacross the entire dynamic range of the device: approximately 500, 1500,8000, 20,000, 30,000, 100,000, 600,000, and 3,000,000 molecules/mL. Atotal of 80 experiments and 29 additional controls were performed, andthe observed concentrations showed excellent agreement with the expectedconcentrations and demonstrated the accuracy of the device performanceover the entire dynamic range (FIG. 16). The experimental resultsconsist of a “digital” pattern of positive and negative wells. At aninput concentration of 1500 molecules/mL (FIG. 3a ), the larger 125 and25 nL wells provide the majority of the information to determine theconcentration. As expected, at a higher concentration of 600,000molecules/mL, positives were found in the smaller 5 and 1 nL wells also(FIG. 16b ), and these smaller wells provide the majority of theinformation used to determine the concentration. Excellent agreement wasfound between the input concentration and the measured concentrationover 4 orders of magnitude (FIG. 16c, d ). In this multivolume design,the 95% confidence interval is narrow at a consistent level over a verylarge range of concentrations: the CI is within 13.8-15% of the expectedvalue from 9500 to 680,000 molecules/mL and within 13.8-17.5% from 5400to 1,700,000 molecules/mL. The experimental data closely tracked thetheoretically predicted CI (FIG. 16d ).

As expected, the largest wells (125 nL) provided the largestcontribution to the overall confidence interval for samples in the10²-10⁴ molecules/mL range while the use of smaller and smaller wellsdown to 1 nL in volume extended the dynamic range with a 95% confidenceinterval above 10⁶ molecules/mL (FIG. 16c, d ). For each concentration,there was excellent agreement among the individual results obtained fromthe wells of different volumes, consistent with the accuracy of theoverall device. This agreement is illustrated for an input concentrationof 30,000 molecules/mL (FIG. 17). At this concentration, the wells ofall volumes provided a reasonable number of positives and negatives forquantification, and we found that the concentration calculated from theresults fell within the 95% confidence intervals for individual volumesof wells (38 of 40 results), and also, the averages from wells of eachvolumes were internally consistent (FIG. 17).

The estimation may, in some embodiments, have a lower detection limit,at a 95% confidence value, in the range of between about 40 molecules/mLsample to about 120 molecules/mL of sample. The methods may suitably becapable of resolving a three-fold difference in viral load of thebiological sample based on an estimate of the level of the one or morenucleic acids.

In other embodiments, the methods include estimating the level,presence, or both of a protein in the biological sample. This estimationmay be effected by (a) contacting the sample with a detection moietycapable of binding to the protein so as to give rise to a population oflabeled proteins, the detection moiety comprising the one or morenucleic acids, (b) disposing the labeled proteins into two or more wellsof different volumes, (c) amplifying the one or more nucleic acids ofthe detection moiety in the two or more wells of different volumes, and(d) correlating an estimated presence or absence of the one or morenucleic acids in the two or more wells of different volumes to a levelof the protein in the biological sample.

As one example, anti-PSA capture antibody coated fluorescent magneticbeads are used to capture the target PSA molecule. The concentration ofPSA may be controlled so there was less than one molecule on one bead. AdsDNA tag is attached to an anti-PSA detection antibody and used assignal probe. After incubation between antibodies and antigen, magneticbeads with captured/labeled PSA are loaded into pL wells with PCRsupermix. Each well contains either one or no bead. After amplification,only wells containing beads are counted. The ratio between “on” wellsand the total number of wells is used to determine the concentration oftarget.

As explained above, relative motion between first and second componentseffects distribution of any contents of the population of first wellsbetween the first population of wells and an additional population ofwells. The relative motion may effect distribution of any contents ofthe population of second wells between the second population of wellsand an additional population of wells.

It should be understood that the methods may include one, two, or moreapplications of relative motion between components. For example, a firstrelative motion (e.g., rotation) may be applied so as to place first andsecond sets of wells into fluid communication with one another. Afterthe contents of the first and second wells contact one another,additional rotation may be applied to place the wells with mixedcontents into fluid communication with another set of wells withdifferent contents, which in turns enables the user to effect processesthat require separate and/or sequential mixing steps of two, three, ormore sample volumes. This may be done, for example, to (1) mix materialsin well A and well B in well A; and then (2) to contact the mixedmaterials in well A with a buffer in well C so as to dilute the contentsof well A. Alternatively, the mixed contents of well A may be contacted(via relative motion of components) with well C such that the contentsof well C may react with the contents of well A (which well included thecontents of well A and well B).

Also provided are kits. The kits suitably include device as set forthelsewhere herein, and also a supply of a reagent selected to participatein nucleic acid amplification. The reagent may be disposed in acontainer adapted to engage with a the conduit of the first component,the conduit of the second component, or both. Such a container may be apipette, a syringe, and the like.

The disclosed devices and kits may also include a device capable ofsupplying or removing heat from the first and second components. Suchdevices include heaters, refrigeration devices, infrared or visiblelight lamps, and the like. The kits may also include a device capable ofcollecting an image of at least some of the first population of wells,the second population of wells, or both.

Amplification Techniques

A non-exclusive listing of suitable isothermal amplification techniquesare provided below. These techniques are illustrative only, and do notlimit the present disclosure.

A first set of suitable isothermal amplification technologies includesNASBA, and RT-RPA. These amplification techniques can operate at 40 deg.C. (a lower temperature preferred for certain POC devices): NASBA(product: RNA), RT-RPA (product: DNA), RT-LAMP using one of LAMP HIV-RNA6-primer sets, transcription-mediated amplification (TMA, 41 deg. C.),helicase dependent amplification (HAD, 65 deg. C.), andstrand-displacement amplification (SDA, 37 deg. C.),

In addition to standard PCR techniques, the disclosed methods anddevices are also compatible with isothermal amplification techniquessuch as loop-mediated amplification (LAMP), Recombinase polymeraseamplification (RPA), nucleic acid sequence based amplification (NASBA),transcription-mediated amplification (TMA), helicase-dependentamplification (HAD), rolling circle amplification (RCA), andstrand-displacement amplification (SDA). The disclosed multivolumedevices can be used to digitize such platforms.

Other isothermal amplification methods are also suitable. Isothermalexponential amplification reaction (EXPAR) may amplify a 10-20 bptrigger oligonucleotide generated from a genomic target more than 106times in less than 10 minutes at 55 deg. C. by repeating cycles ofpolymerase and endonuclease activity, and has been coupled withDNA-functionalized gold nanospheres for the detection of herpes simplexvirus. Isothermal and chimeric primer-initiated amplification of nucleicacids (ICANs) amplify target DNA at 55 deg. C. using a pair of50-DNA-RNA-30 primers and the activity of RNase H and strand displacingpolymerase.

Signal-mediated amplification of RNA technology (SMART) produces copiesof an RNA signal at 41 deg. C. in the presence of an RNA or DNA targetby way of the three-way junction formed between the target and twoprobes, one of which contains the RNA signal sequence and a T7 promotersequence for T7 RNA polymerase. The single stranded RNA product may bedetected by hybridization-based methods and because the signal isindependent of the target, SMART may be used for detection of differenttarget sequences. Cyclic enzymatic amplification method (CEAM) detectsnucleic acids in the picomolar range in less than 20 minutes at 37 deg.C. using a displacing probe and Exonuclease III (Exo III) to generateamplification of fluorescent signal in the presence of a target.Isothermal target and signaling probe amplification (iTPA) combines theprinciple of ICAN and the inner-outer probe concept of LAMP along withfluorescence resonance energy transfer cycling probe technology (FRETCPT) for simultaneous target and signal amplification in 90 minutes at60 deg. C., and has been shown to detect Chlamydia trachomatis at singlecopy level.

Other suitable amplification methods include ligase chain reaction(LCR); amplification methods based on the use of Q-beta replicase ortemplate-dependent polymerase; helicase-dependent isothermalamplification; strand displacement amplification (SDA); thermophilic SDAnucleic acid sequence based amplification (3SR or NASBA) andtranscription-associated amplification (TAA).

Non-limiting examples of PCR amplification methods include standard PCR,AFLP-PCR, Allele-specific PCR, Alu-PCR, Asymmetric PCR, BiasedAllele-Specific (BAS) Amplification, Colony PCR, Hot start PCR, InversePCR (IPCR), In situ PCR (ISH), Intersequence-specific PCR (ISSR-PCR),Long PCR, Multiplex PCR, Nested PCR, Quantitative PCR, ReverseTranscription PCR (RT-PCR), Real Time PCR, Single cell PCR, Solid phasePCR, Universal Size-Specific (USS-PCR), branched-DNA technology, and thelike

Further amplification techniques are described below. Each of thesetechniques is suitably performed by the disclosed devices and methods.Allele-specific PCR is a diagnostic or cloning technique based onsingle-nucleotide polymorphisms (SNPs) (single-base differences in DNA).It requires some knowledge of a DNA sequence, including differencesbetween alleles, and uses primers whose 3′ ends encompass the SNP. PCRamplification may be less efficient in the presence of a mismatchbetween template and primer, so successful amplification with anSNP-specific primer signals presence of the specific SNP in a sequence.

Assembly PCR or Polymerase Cycling Assembly (PCA) is an artificialsynthesis of long DNA sequences by performing PCR on a pool of longoligonucleotides with short overlapping segments. The oligonucleotidesalternate between sense and antisense directions, and the overlappingsegments determine the order of the PCR fragments, thereby selectivelyproducing the final long DNA product.

Asymmetric PCR preferentially amplifies one DNA strand in adouble-stranded DNA template. It is used in sequencing and hybridizationprobing where amplification of only one of the two complementary strandsis required. PCR is carried out as usual, but with a great excess of theprimer for the strand targeted for amplification. Because of the slow(arithmetic) amplification later in the reaction after the limitingprimer has been used up, extra cycles of PCR are required. A recentmodification on this process, known as Linear-After-The-Exponential-PCR(LATE-PCR), uses a limiting primer with a higher melting temperature(Tm) than the excess primer to maintain reaction efficiency as thelimiting primer concentration decreases mid-reaction.

Helicase-dependent amplification is similar to traditional PCR, but usesa constant temperature rather than cycling through denaturation andannealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, isused in place of thermal denaturation.

Hot start PCR is a technique that reduces non-specific amplificationduring the initial set up stages of the PCR. It may be performedmanually by heating the reaction components to the denaturationtemperature (e.g., 95° C.) before adding the polymerase. Specializedenzyme systems have been developed that inhibit the polymerase'sactivity at ambient temperature, either by the binding of an antibody orby the presence of covalently bound inhibitors that dissociate onlyafter a high-temperature activation step. Hot-start/cold-finish PCR isachieved with new hybrid polymerases that are inactive at ambienttemperature and are activated at elongation temperature.

Inter-sequence-specific PCR (ISSR) is a PCR method for DNAfingerprinting that amplifies regions between simple sequence repeats toproduce a unique fingerprint of amplified fragment lengths.

Inverse PCR is commonly used to identify the flanking sequences aroundgenomic inserts. It involves a series of DNA digestions andself-ligation, resulting in known sequences at either end of the unknownsequence.

Ligation-mediated PCR: uses small DNA linkers ligated to the DNA ofinterest and multiple primers annealing to the DNA linkers; it has beenused for DNA sequencing, genome walking, and DNA footprinting.

Methylation-specific PCR (MSP) is used to detect methylation of CpGislands in genomic DNA. DNA is first treated with sodium bisulfite,which converts unmethylated cytosine bases to uracil, which is in turnrecognized by PCR primers as thymine. Two PCRs are then carried out onthe modified DNA, using primer sets identical except at any CpG islandswithin the primer sequences. At these points, one primer set recognizesDNA with cytosines to amplify methylated DNA, and one set recognizes DNAwith uracil or thymine to amplify unmethylated DNA. MSP using qPCR canalso be performed to obtain quantitative rather than qualitativeinformation about methylation.

Miniprimer PCR uses a thermostable polymerase (S-Tbr) that can extendfrom short primers (“smalligos”) as short as 9 or 10 nucleotides. Thismethod permits PCR targeting to smaller primer binding regions, and isused to amplify conserved DNA sequences, such as the 16S (or eukaryotic18S) rRNA gene.

Multiplex Ligation-dependent Probe Amplification (MLPA) permits multipletargets to be amplified with only a single primer pair, as distinct frommultiplex-PCR.

Multiplex-PCR: consists of multiple primer sets within a single PCRmixture to produce amplicons of varying sizes that are specific todifferent DNA sequences. By targeting multiple genes at once, additionalinformation may be gained from a single test-run that otherwise wouldrequire several times the reagents and more time to perform. Annealingtemperatures for each of the primer sets may be optimized to workcorrectly within a single reaction, and amplicon sizes. That is, theirbase pair length may be different enough to form distinct bands whenvisualized by gel electrophoresis.

Nested PCR: increases the specificity of DNA amplification, by reducingbackground due to non-specific amplification of DNA. Two sets of primersare used in two successive PCRs. In the first reaction, one pair ofprimers is used to generate DNA products, which besides the intendedtarget, may still consist of non-specifically amplified DNA fragments.The product(s) are then used in a second PCR with a set of primers whosebinding sites are completely or partially different from and located 3′of each of the primers used in the first reaction.

Overlap-extension PCR or Splicing by overlap extension (SOE): a geneticengineering technique that is used to splice together two or more DNAfragments that contain complementary sequences. The technique is used tojoin DNA pieces containing genes, regulatory sequences, or mutations;the technique enables creation of specific and long DNA constructs.

Quantitative PCR (Q-PCR): used to measure the quantity of a PCR product(commonly in real-time). It quantitatively measures starting amounts ofDNA, cDNA, or RNA. Q-PCR is commonly used to determine whether a DNAsequence is present in a sample and the number of its copies in thesample. Quantitative real-time PCR can have a high degree of precision.QRT-PCR (or QF-PCR) methods use fluorescent dyes, such as Sybr Green,EvaGreen or fluorophore-containing DNA probes, such as TaqMan, tomeasure the amount of amplified product in real time. It is alsosometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR orRTQ-PCR are more appropriate contractions, as RT-PCR commonly refers toreverse transcription PCR (see below), often used in conjunction withQ-PCR.

Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reversetranscriptase reverse transcribes RNA into cDNA, which is then amplifiedby PCR. RT-PCR is widely used in expression profiling, to determine theexpression of a gene or to identify the sequence of an RNA transcript,including transcription start and termination sites. If the genomic DNAsequence of a gene is known, RT-PCR can be used to map the location ofexons and introns in the gene. The 5′ end of a gene (corresponding tothe transcription start site) is typically identified by RACE-PCR (RapidAmplification of cDNA Ends).

Solid Phase PCR: encompasses multiple meanings, including PolonyAmplification (where PCR colonies are derived in a gel matrix, forexample), Bridge PCR (primers are covalently linked to a solid-supportsurface), conventional Solid Phase PCR (where Asymmetric PCR is appliedin the presence of solid support bearing primer with sequence matchingone of the aqueous primers) and Enhanced Solid Phase PCR (whereconventional Solid Phase PCR can be improved by employing high Tm andnested solid support primer with optional application of a thermal stepto favor solid support priming).

Thermal asymmetric interlaced PCR (TAIL-PCR) may be useful for isolationof an unknown sequence flanking a known sequence. Within the knownsequence, TAIL-PCR uses a nested pair of primers with differingannealing temperatures; a degenerate primer is used to amplify in theother direction from the unknown sequence.

Touchdown PCR (Step-down PCR) is a variant of PCR that aims to reducenonspecific background by gradually lowering the annealing temperatureas PCR cycling progresses. The annealing temperature at the initialcycles is usually a few degrees (3-5° C.) above the Tm of the primersused, while at the later cycles, it is a few degrees (3-5° C.) below theprimer Tm. The higher temperatures give greater specificity for primerbinding, and the lower temperatures permit more efficient amplificationfrom the specific products formed during the initial cycles.

PAN-AC uses isothermal conditions for amplification, and may be used inliving cells.

Universal Fast Walking is useful for genome walking and geneticfingerprinting using a more specific two-sided PCR than conventionalone-sided approaches (using only one gene-specific primer and onegeneral primer) by virtue of a mechanism involving lariat structureformation. Streamlined derivatives of UFW are LaNe RAGE(lariat-dependent nested PCR for rapid amplification of genomic DNAends), 5′ RACE LaNe, and 3′ RACE LaNe.

COLD-PCR (co-amplification at lower denaturation temperature-PCR) is amodified Polymerase Chain Reaction (PCR) protocol that enriches variantalleles from a mixture of wildtype and mutation-containing DNA.

Another alternative isothermal amplification and detection method thatis isothermal in nature is described athttp://www.invaderchemistry.com/(Invader Chemistry™) This method may beperformed by the disclosed devices and methods. Another alternativeamplification technique (so-called qPCR) is disclosed by MNAzyme(http://www.speedx.com.au/MNAzymeqPCR.html), which technique is alsosuitable for the presently disclosed devices and methods.

One may also effect amplification based on nucleic acid circuits (whichcircuits may be enzyme-free). The following references (all of which areincorporated herein by reference in their entireties) describe exemplarycircuits; all of the following are suitable for use in the discloseddevices and methods: Li et al., “Rational, modular adaptation ofenzyme-free DNA circuits to multiple detection methods,” Nucl. AcidsRes. (2011) doi: 10.1093/nar/gkr504; Seelig et al., “Enzyme-Free NucleicAcid Logic Circuits,” Science (Dec. 8, 2006), 1585-1588; Genot et al,“Remote Toehold: A Mechanism for Flexible Control of DNA HybridizationKinetics,” JACS 2011, 133 (7), pp 2177-2182; Choi et al., “Programmablein situ amplification for multiplexed imaging of mRNA expression,”Nature Biotechnol, 28:1208-1212, 2010; Benner, Steven A., and A. MichaelSismour. “Synthetic Biology.” Nat Rev Genet 6, no. 7 (2005): 533-543;Dirks, R. M., and N. A. Pierce. “Triggered Amplification byHybridization Chain Reaction.” Proceedings of the National Academy ofSciences of the United States of America 101, no. 43 (2004): 15275;Graugnard, E., A. Cox, J. Lee, C. Jorcyk, B. Yurke, and W. L. Hughes.“Kinetics of DNA and Rna Hybridization in Serum and Serum-Sds.”Nanotechnology, IEEE Transactions on 9, no. 5 (2010): 603-609; Li,Bingling, Andrew D. Ellington, and Xi Chen. “Rational, ModularAdaptation of Enzyme-Free DNA Circuits to Multiple Detection Methods.”Nucleic Acids Research, (2011); Li, Q., G. Luan, Q. Guo, and J. Liang.“A New Class of Homogeneous Nucleic Acid Probes Based on SpecificDisplacement Hybridization.” Nucleic Acids Research 30, no. 2 (2002):e5-e5; Picuri, J. M., B. M. Frezza, and M. R. Ghadiri. “UniversalTranslators for Nucleic Acid Diagnosis.” Journal of the AmericanChemical Society 131, no. 26 (2009): 9368-9377; Qian, Lulu, and ErikWinfree. “Scaling up Digital Circuit Computation with DNA StrandDisplacement Cascades.” Science 332, no. 6034 (2011): 1196-1201;Tsongalis, G. J. “Branched DNA Technology in Molecular Diagnostics.”American journal of clinical pathology 126, no. 3 (2006): 448-453; VanNess, Jeffrey, Lori K. Van Ness, and David J. Galas. “IsothermalReactions for the Amplification of Oligonucleotides.” Proceedings of theNational Academy of Sciences 100, no. 8 (2003): 4504-4509; Yin, Peng,Harry M. T. Choi, Colby R. Calvert, and Niles A. Pierce. “ProgrammingBiomolecular Self-Assembly Pathways.” Nature 451, no. 7176 (2008):318-322; Zhang, D. Y., and E. Winfree. “Control of DNA StrandDisplacement Kinetics Using Toehold Exchange.” Journal of the AmericanChemical Society 131, no. 47 (2009): 17303-17314; Zhang, David Yu,Andrew J. Turberfield, Bernard Yurke, and Erik Winfree. “EngineeringEntropy-Driven Reactions and Networks Catalyzed by DNA.” Science 318,no. 5853 (2007): 1121-1125; Zhang, Z., D. Zeng, H. Ma, G. Feng, J. Hu,L. He, C. Li, and C. Fan. “A DNA-Origami Chip Platform for Label-FreeSNP Genotyping Using Toehold-Mediated Strand Displacement.” Small 6, no.17 (2010): 1854-1858.

It should also be understood that the present disclosure is not limitedto application to molecules, as the disclosed devices and methods may beapplied to organisms (e.g., those described in paragraph 0133 ofpriority application PCT/US2010/028316 and also elsewhere in thatapplication), single cells, single biological particles (e.g.,bacteria), single vesicles, single exosomes, single viruses, singlespores, lipoprotein particles, and the like, and single non-biologicalparticles. One exemplary analysis of lipoprotein particles may be foundat www.liposcience.com. Furthermore, it should also be understood thatthe disclosed devices and methods may be applied to stochasticconfinement (described in, for example, “Stochastic Confinement toDetect, Manipulate, And Utilize Molecules and Organisms,” patentapplication PCT/US2008/071374), and reactions and manipulations ofstochastically confined objects. As one non-limiting example, biologicalsamples may be assessed for the presence or level of certain bacteria,such as those organisms that serve as markers for bacterial vaginosis.This assessment may be performed by amplifying nucleic acids that may bepresent in the sample and correlating the levels of those nucleic acidsto the presence or absence of the marker organisms. One exemplaryanalysis is found at http://www.viromed.com/client/cats/BV %20LAB.pdf.

It should be understood that “nucleic acid” is not limited to DNA.“Nucleic acid” should be understood as referring to RNA and/or a DNA.Exemplary RNAs include, but are not limited to, mRNAs, tRNAs, snRNAs,rRNAs, retroviruses, small non-coding RNA, microRNAs, ploysomal RNAs,pre-mRNAs, intromic RNA, and viral RNA. Exemplary DNAs include, but arenot limited to, genomic DNA, plasmid DNA, phage DNA, nucleolar DNA,mitochondrial DNA, chloroplast DNA, cDNA, synthetic DNA, yeastartificial chromosomal DNA, bacterial artificial chromosomal DNA, otherextrachromosomal DNA, and primer extension products.

In some embodiments, a nucleic acid comprises PNA and/or LNA (lockednucleic acid). In still other embodiments, a nucleic acid comprises oneor more aptamers that can be in the form of single stranded DNA, RNA, ormodified nucleic acids. Nucleic acid aptamers may be single stranded ordouble stranded. In some embodiments, nucleic acid contains nucleotideswith modified synthetic or unnatural bases, including any modificationto the base, sugar or backbone. Further information is found in U.S.Pat. No. 7,790,385 and also in United States patent applicationpublications US2008/0032310, US2008/0050721, and US2005/0089864, all ofwhich are incorporated herein by reference in their entireties for anyand all purposes.

It should also be understood that in some embodiments, the discloseddevices and methods provide for detection of target molecules with orwithout quantification (or estimated quantification) of the targetmolecules. Accordingly, it is not necessary for a user to estimate theconcentration or level of a target in a sample; the disclosed devicesand methods may be used to detect the presence of a target in a yes/nofashion. This is especially useful in applications where the user maydesire only to know whether a particular target (e.g., a virus) ispresent; in such cases, the precise level of the target is of lesserimportance.

Such detection can be carried out by physical, chemical, and biologicalreactions, such as hybridization, nucleic acid amplification,immunoassays, and enzymatic reaction. In some embodiments, thisdetection method can be used for qualitative analysis of one or moretarget molecules. As describe above, in some embodiments material may,after a reaction, processing, or even a detection step, be transferredto another device, or even transferred to another region of the samedevice. In some embodiments, recovered material may be removed from thedevice. In some embodiments, material after detection may be recoveredfrom device and further analyzed. Such recovery may be carried out, insome embodiments, by accessing individual wells of a device, e.g., by apipettor. In some embodiments, recovery may be achieved by combiningmaterial from multiple wells, for example by actuating a device to theloading position and using a carrier fluid to expel the material fromthe device.

EXEMPLARY EMBODIMENTS

The following illustrates an exemplary embodiment of the discloseddevices. The embodiment comprises a rotationally-configured device forquantifying RNA with a large dynamic range by using multivolume digitalRT-PCR (MV digital RT-PCR).

Quantitative detection of RNA provides valuable information for study ofgene expression, and has the potential to improve evaluation of diseases(including stroke, leukemia, and prostate cancer), analysis of graftrejection in transplantation, and vaccine development. Quantification ofviral RNA has also become useful for monitoring the progression of viralinfection and efficacy of applied treatment.

One such instance is in the treatment of HIV. More than 33 millionpeople worldwide are living with HIV, and a large number of them are indeveloping countries and resource-limited areas. First-lineantiretroviral treatment is becoming widely available, and it greatlyincreases both the duration and quality of life of HIV patients.However, this first-line treatment can fail as the virus mutates anddevelops drug resistance. In order to stop the global spread of drugresistance and provide proper treatment for patients, it is critical toevaluate the HIV viral load at regular intervals (every 3 to 4 months)after initial treatment is shown to be effective. HIV viral loadmeasurement is a particularly useful tool for diagnosing and evaluatingthe status of HIV infection in children under age 18 months.

The hepatitis C virus (HCV) infection is also a significant globalhealthcare burden, as it has been identified as one of the major causesof liver disease and is one of the most common co-infections of HIV. HCVviral load may also need to be monitored to determine the effectivenessof treatment.

The viral load for chronic HCV can range from about 50,000 to about 5million international units per mL (IU/mL), while for patientsresponding to antiviral treatment the load will be lower. Successfultreatment should result in essentially undetectable levels of HCV viralRNA, and the assessment of such treatment may require HCV viral loadmeasurements capable of a wide dynamic range.

As explained previously, real time quantitative RT-PCR is one standardfor monitoring viral load for HIV, HCV, and other viral infections.However, this test is cost-prohibitive under resource-limited settingsand usually requires multiple instruments, highly skilled technicians,and isolated rooms to prevent contamination. Moreover, the efficiency ofRT-PCR, the quality of sample and selection of targets, and the methodsfor interpretation of the data present concerns for the accuracy ofquantifying RNA using RT-PCR.

While a dipstick device has been developed that providessemiquantitative measurements of HIV viral load after amplification inresource limited settings, no quantitative test exists to resolve a3-fold (0.5 log¹⁰) change in HIV RNA viral load, which is considered tobe clinically significant. Digital PCR is one method that performsquantitative analysis of nucleic acids by detecting single molecule ofDNA or RNA and can provide an absolute count of the nucleic acid copynumber with potentially higher accuracy compared to real time PCR.Existing applications of digital PCT, however, require significant skilland resource commitments.

The exemplary, disclosed devices present a microfluidic platform thatcan manipulate liquid samples from picoliter-to-microliter scales byrelative movement of different plates without the need for complexcontrol systems. These devices may be used for multiplex PCR, digitalPCR, and digital isothermal amplification (RPA).

In place of using wells of uniform size, using wells of multiple volumesto achieve the same dynamic range can reduce the total number of wellsand increase the spacing among wells to simplify imaging and downstreamanalysis. A mathematical approach for experimental design andstatistical analysis for multivolume digital PCR (MV digital PCR) hasbeen characterized using DNA in Kreutz et al., Anal. Chem. 2011, DOI10.1021/ac201658s, incorporated herein by reference for all purposes.

A disclosed devices was applied, as set forth below, to quantitativeanalysis of RNA with large dynamic range by MV digital RT-PCR. Thisdevice was characterized with a serial dilution of a synthetic controlRNA molecule of 906 nucleotides (906 nt). Also described is a seconddesign of the platform that maintains a large dynamic range for fivesamples simultaneously, allowing for multiplexed experiments. Thissystem was validated by using HCV control viral RNA and HIV viral RNAtogether with internal controls. The system also displayed the use ofmultivolume designs to quantify HIV viral load at a large dynamic rangeby quantifying purified HIV viral RNA from clinical patients' samples.

Results

First characterized was a multivolume digital device (Design 1, Table 1,FIG. 11) with a large dynamic range suitable for viral load testing.This device contained four different volumes (1 nL, 5 nL, 25 nL, 125 nL)with 160 wells each (FIG. 1A) for a theoretical dynamic range (lowerdynamic range, LDR, to upper limit of quantification, ULQ) of 5.2×10² to4.0×10⁶ molecules/mL at 3-fold resolution and a lower detection limit(LDL) of 1.2×10² molecules/mL in the final RT-PCR mixture. The LDRcorresponds to the lowest concentration that can be resolved from a 3-or 5-fold higher concentration; the ULQ is the concentration that has a95% chance of generating at least one negative well and is equal to theconcentration calculated from three negative wells; the LDL is theconcentration that has a 95% chance of generating at least one positivewell and is equal to the concentration calculated from three positivewells.

Continuous fluidic paths are generated by partially overlapping thewells in the top plate and the wells in the bottom plate (e.g., FIG. 1B,FIG. 1E; FIG. 15). The design of this device follows the generalprinciples of dead-end filling for complete filling of aqueous reagents(FIG. 1C,F). After complete loading, the top plate is slipped (rotated)clockwise by ˜8° to break the fluidic path and overlay the wells filledwith solution with the wells in the facing plate used to control thermalexpansion (FIG. 1D, FIG. 1G). The device is then placed on a flat insitu adaptor for thermal cycling.

The theory for design and analysis of this multivolume device aredescribed in detail and validated by using digital PCR for DNA. Briefly,concentrations were calculated based on Most Probable Number (MPN)theory by combining the results from each volume (i=1, 2, 3, 4) in thefirst equation below and solving for λ (concentration, molecules/mL),where n_(i) is the total number of wells at each volume, b_(i) is thenumber of negative wells at that volume, and v_(i) is the well volume(mL). Combining results allows for more precise identification of the“most probable” concentration and also improves the confidence interval.To find the confidence interval, the standard deviation, σ, for ln(λ) isdetermined using the second equation below, which was derived based onthe Fisher information.

${\sum\limits_{i = 1}^{m}{n_{i}v_{i}}} = {\sum\limits_{i = 1}^{m}\frac{\left( {n_{i} - b_{i}} \right)v_{i}}{\left( {1 - e^{{- v_{i}}\lambda}} \right)}}$$\sigma = \frac{1}{\sqrt{\lambda^{2}{\sum\frac{v_{i}^{2}n_{i}}{e^{v_{i}\lambda} - 1}}}}$

To validate the performance of the multivolume device with RNA, digitalRT-PCR ws performed using a six order—of magnitude serial dilution ofsynthetic control RNA template (906 nt). This control RNA wassynthesized from a control plasmid and purified by using a commercialpurification kit. The concentration of the stock solution of control RNAwas measured spectrophotometrically by a NanoDrop™ device to be ˜1.8ng/μL, corresponding to ˜4.1×10¹² molecules/mL, which may contain somebackground signal.

Using the device and through statistical analysis of all MV digitalRT-PCR results (FIG. 3), a nominal real concentration of the control RNAin solution was obtained, 2.2×10¹² molecules/mL, which value was used asthe true concentration of all MV digital RT-PCR results reported in FIG.3. A RT-PCR master mix containing EvaGreen Super-Mix, RT-transcriptase,bovine serum albumin (BSA), and primers was mixed with the RNA templatesolution. EvaGreen, an intercalating dye, was used for end-pointfluorescent imaging after thermal cycling (FIG. 2).

FIG. 1 illustrates a rotational multivolume device (well volumes: 1 nL,5 nL, 25 nL, 125 nL). (A) Bright field image of the rotational deviceafter slipping to form isolated compartments, shown next to a U.S.quarter. (B-D) Schematics and (E-G) bright field microphotograph show(B, E) the assembled rotational device. (C, F) The device filled withfood dye after dead-end filling. (D, G) The device after rotationalslipping: 640 aqueous droplets of four different volumes (160 wells withvolumes of 1 nL, 5 nL, 25 nL, 125 nL each) were formed simultaneously.In the schematics, dotted lines indicate features in the top plate, andblack solid lines represent the features in the bottom plate.

FIG. 2 shows end-point fluorescence images of multivolume digital RTPCRperformed on a rotational device for synthetic RNA template at fivedifferent concentrations. (A) Control, containing no RNA template. (B-F)Serial dilution of 906 nt RNA template from 2.2×10² to 2.2×10⁶molecules/mL in the RT-PCR mix.

No false positives were observed after amplification in four negativecontrol experiments, as there was no significant increase of fluorescentintensity in wells (FIG. 2A). As the concentration of RNA templateincreased (the dilution factor decreased), the fraction of positivewells in each set of individual volumes was counted and theconcentration of template in the RT-PCR mix was calculated as describedabove (FIG. 2B-F). The glass device was reused after being thoroughlycleaned with piranha acid (3:1 sulfuric acid:hydrogen peroxide), plasmacleaned, and resilanized with dichlorodimethylsilane. Four to fiveexperiments were performed for each concentration of template, and thecalculated concentration of template RNA showed good agreement withinthe expected statistical distribution at each concentration and scaledlinearly with the expected concentration (FIG. 3A). The results at theconcentrations of 2.2×10⁶, 2.2×10⁵, 2.2×10⁴, 2.2×10³, 2.2×10², and7.3×10¹ molecules/mL in the RT-PCR mix were used to estimate an initialstock concentration of control RNA of approximately 2.2×10¹²molecules/mL. The experimental results across the concentrations agreewell with the theoretically predicted distribution (FIG. 3A,B). Of the26 experiments, 19 fall within the 95% confidence interval and 22 fallwithin the 99% confidence interval.

FIG. 3 presents the performance of digital RT-PCR with synthetic RNAtemplate on the multivolume device over a 4 log₁₀ dynamic range,comparing the expected concentration of RNA in RT-PCR mix to (A) theobserved concentration, and (B) the ratio of the observed/expectedconcentration. Individual experimental results (crosses) and averageresults (crosses) for concentration were plotted against the dilutionlevel of the RNA stock solution. Four to five experiments were performedat each concentration, and some experimental results are overlapping.The experimental results show a linear relationship with the dilutionlevel and fit within the expected distribution. The experimental resultswere used to estimate an initial stock concentration, whose distributionwas then fit to the dilution level to provide the expected value (blackcurve) and 95% confidence interval (gray curves).

Over the dynamic range of the device, the contribution of wells withdifferent volumes to the calculated concentration varies, approximatedin FIG. 4A. As the concentration of control RNA template increases (thedilution decreases), the major contribution to the calculated finalconcentration shifts from wells of large volume (125 nL) to wells ofmedium volume (25 nL and 5 nL) and then to wells of small volume (1 nL).The percent that the result from each volume contributes to σ serves asan estimate of the relative contribution of that volume to theconcentration determined by all volumes on the entire chip. In FIG. 4A,the data bars for the 125 nL volume are for (left to right) 2.2×10²molecules/mL, 2.2×10³ molecules/mL, and 2.2×10⁴ molecules/mL. For 25 nL,data bars are (left to right) 2.2×10² molecules/mL, 2.2×10³molecules/mL, 2.2×10⁴ molecules/mL, 2.2×10⁵ molecules/mL, and 2.2×10⁶molecules/mL. For 5 nL volumes, the data bars are (left to right)2.2×10² molecules/mL, 2.2×10³ molecules/mL, 2.2×10⁴ molecules/mL,2.2×10⁵ molecules/mL, and 2.2×10⁶ molecules/mL. For 1 nL volumes, thedata bars are (left to right) 2.2×10⁴ molecules/mL, 2.2×10⁵molecules/mL, and 2.2×10⁶ molecules/mL. The concentration calculatedfrom analysis of positive and negative wells of each of the volumes onthe individual device was selfconsistent and was consistent with thecalculated concentration determined by combining all wells withdifferent volumes (FIG. 4B). This result indicates that multivolumedigital approach is fully compatible with analysis of RNA by RT-PCR.

FIG. 4A shows that for each dilution, the approximate contributions ofthe results from each well volume toward calculating the finalconcentration were calculated based on the contributions of each volumeto the standard deviation, σ. FIG. 4(B), showing the concentration ofRNA template calculated from the overall chip (combining all wellvolumes, solid bars) and individual volumes (patterned bars) isself-consistent on the MV digital RT-PCR device. Four experiments wereperformed with 2.2×10⁴ molecules/mL of control RNA template (906 nt) inthe RT-PCR mix.

To illustrate incorporation of multiplexing into the device whilemaintaining the high dynamic range, the design of the multivolume devicewas modified by adding two additional volumes (FIG. 11, Design 2A): 0.2nL (160 wells) and 625 nL (80 wells). When the rotational chip is splitinto five sections to quantify five different analytes, the 0.2 nL wellsextend the upper limit of quantification with 3-fold resolution to1.2×10⁷ molecules/mL in the RT-PCR mix, and the 625 nL wells maintain areasonable lower detection limit of 2.0×10² molecules/mL and lowerdynamic range with 3-fold resolution at 1.8×10³ molecules/mL in theRT-PCR mix (FIG. 5A). The higher upper limit of quantification isrequired to quantify HCV viral RNA, and the lower dynamic range andlower detection limit are required for the HIV viral load test. Fivedifferent solutions can be introduced into the device simultaneously(FIG. 5A) for multiplexed analysis.

As HCV is one of the most common co-infections for HIV patients,validation was performed on the multiplexed device with a five-plexpanel: measurement of HIV viral RNA, measurement of HCV control viralRNA, a negative control for HIV, a negative control for HCV, andmeasurement of 906 nt control RNA in HCV sample for quantification ofsample recovery rate (FIG. 5B,C). The 906 nt control RNA was the sameone characterized by using digital RT-PCR on the device (design 1; seeFIGS. 1 and 11). HIV viral RNA was purified from an archived sample ofplasma containing HIV (viral RNA estimated to be ˜1.5×10⁶ molecules/mL)from a de-identified patient sample, and HCV control viral RNA waspurified from a commercial sample containing control HCV virus (25million IU/mL, OptiQuant-S HCV Quantification Panel, Acrometrix) usingthe iPrep purification instrument, as described elsewhere herein. As thefinal elution volume of purified nucleic acid is generally smaller thanthe starting volume of plasma, there is a concentrating effect on viralRNA after sample purification. To characterize this concentratingeffect, the 906 nt control RNA with known concentration was added to thelysed plasma and was quantified again after sample preparation. Theratio of the concentration of 906 nt control RNA after/before samplepreparation is defined as the concentrating factor. The concentratingfactors after sample purification were approximately 6.6 for HIV viralRNA and approximately 4.5 for HCV control viral RNA. Primers for HIV andHCV were selected. Only one pair of primers was added to each sample,and the experiment was repeated six times. In those six experiments, nofalse positives were observed in either HIV or HCV negative controlpanels after thermal cycling, and no crosscontamination was observedamong different panels. From these six experiments, the averagecalculated concentration of HIV viral RNA after purification was 7.9×10⁶molecules/mL with standard deviation of 2.5×10⁶ molecules/mL,corresponding to 1.2×10⁶ molecules/mL with standard deviation of 3.7×10⁵molecules/mL in the original plasma sample. The average concentration ofHCV control viral RNA after purification was 1.0×10⁸ molecules/mL withstandard deviation of 4.4×10⁷ molecules/mL, corresponding to 2.3×10⁷molecules/mL with standard deviation of 9.7×10⁶ molecules/mL in theoriginal control plasma sample. (Additional information may be found in“Multiplexed Quantification of Nucleic Acids,” Shen et al., JACS 2011.)

There is no universal conversion factor from international units to copynumber for HCV viral load; it is a value that depends on the detectionplatform, including the protocols and equipment used. Because the HCVconcentration in the original commercial sample was stated to be 2.5×10⁷IU/mL, the conversion factor from international units to copy number forHCV viral load in the test is approximately 0.9. The same conversionnumber (0.9) was published for the Roche Amplicor HCV Monitor v2.0 testwhen using a manual purification procedure.

FIG. 5 illustrates a device for multiplexed, multivolume digital RT-PCRwith high dynamic range. (A) A photograph of a multiplex device for upto five samples corresponding to designs 2A and 2B in Table 1 (FIG. 11)with a total of 80 wells of 625 nL, 160 wells of 125 nL, 160 wells of 25nL, 160 wells of 5 nL, 160 wells of 1 nL, and 160 wells of 0.2 nL. (B)Fluorescent photograph of a multiplexed digital RT-PCR detection panel:(I) measurement of internal control of 906 nt RNA template in HCVsample; (II) HCV control viral RNA measurement; (III) negative controlfor HIV (HIV primers with no loaded HIV RNA template); (IV) HIV viralRNA measurement; (V) negative control for HCV (HCV primers with noloaded HCV RNA template). Inset shows an amplified area from HCV viralload test.

FIG. 6 shows multivolume digital RT-PCR for quantification of HIV viralload in two patients' samples. Input concentration was calculated from asingle clinical measurement for each patient using the Roche CAP/CTMv2.0 system and was assumed to be the true concentration. Eachconcentration was measured at least four times, and each individualexperiment is plotted as single point on the graph. The black solid lineis the predicted concentration based on the assumption that the clinicalmeasurement gave a true concentration. The gray solid lines werecalculated using MPN theory and represent the 95% confidence intervalfor the predicted concentration.

The tabular summary in FIG. 9 present the detection and quantificationdata and dynamic range for the two designs investigated here. Withoutbeing bound to any single theory, the dynamic range of design 1 can beeasily extended by adding a set of wells smaller than 1 nL in volume anda set of wells larger than 125 nL in volume. Therefore, if a largerdynamic range is required, the multiplexed design (Design 2A, FIG. 11)may be used for a single sample (Design 2B, FIG. 11). When using theentire chip for one sample, the 160 smallest wells (0.2 nL in volume)extend the upper limit of quantification with 3-fold resolution to2.0×10⁷ molecules/mL in the RT-PCR mix and the 80 largest wells (625 nLin volume) extend the lower detection limit to 40 molecules/mL and lowerdynamic range with 3-fold resolution to 1.7×10² molecules/mL in theRT-PCR mix (Table 1, Design 2B, FIG. 11). This large dynamic range isuseful for quantification of viral load.

A RT-PCR mix containing an HIV viral RNA sample (prepared as describedabove and then serially diluted) with an expected concentration of 51molecules/mL was used to test the lower detection limit of design 2B(FIG. 11). Three negative control experiments were performed (withoutHIV viral RNA) in parallel, and no false positives were observed. Sixexperiments were performed to quantify the viral RNA concentration (seeFIG. 7), and the average calculated HIV viral RNA concentration in theRT-PCR mix was 70 molecules/mL with standard deviation of 20molecules/mL, corresponding to 32 molecules/mL with standard deviationof 9 molecules/mL in the original plasma sample.

To further validate the feasibility of using a rotational multivolumedevice to quantify HIV viral load, Design 1 (FIG. 11) was used tomeasure HIV viral RNA purified from two archived samples of HIV-infectedblood plasma from two different anonymous patients. The HIV viral RNAfrom each patient sample was extracted and purified automatically usingthe iPrep purification instrument, and concentrating factors of 7.1 and6.6 were achieved for the two different patient samples. Each patientsample of purified HIV viral RNA was serially diluted and characterizedby MV digital RT-PCR on the device using previously published primers,and each experiment was repeated at least four times (FIG. 6). The sameplasma samples were characterized in a single experiment using the RocheCOBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (CAP/CTM v2.0) accordingto the manufacturer's recommendation, and these values were treated asthe standard for characterization. The data from device wereself-consistent for both patients (FIG. 6). Three negative controlexperiments using the same primers but no HIV template did not showfalse positive, as no increase of fluorescent intensity was observed(see FIG. 10).

For patient 1, the results (FIG. 6, crosses) were on averageapproximately 40% lower than that predicted by the single-pointmeasurement of the HIV viral load using Roche CAP/CTM v2.0 (see FIG. 7).There were differences in the test designs: while the present experimenttargets a single LTR region of HIV RNA, the Roche CAP/CTM v2.0 testincludes two HIV sequences: one in gag and another in LTR region.Further, the two tests use different detection methods (EvaGreen in thepresent experiment vs TaqMan probes in the Roche CAP/CTM v2.0 test) anddifferent internal controls. For patient 2, excellent agreement with theRoche clinical measurement was observed over the entire range (FIG. 6,plus marks; see also FIG. 10 (tabular summary). Without being bound toany single theory, this difference in agreement between the two methodsfor the two samples is not surprising, given that each patient has aunique HIV viral genome, and the primers, internal controls, anddetection method used in one method may be better suited to detect onepatient's viral genome than another's. Overall, taking intoconsideration the concentrating effect during sample preparation, thelowest concentration of serially diluted HIV viral RNA detected on thedevice corresponded to 37 molecules/mL in the patient plasma, and thehighest concentration corresponded to 1.7 million molecules/mL in thepatient plasma.

Results Summary

Motivated by the problem of quantifying viral load under point-of-careand resource-limited settings, here is shown successful testing of theapplicability of multivolume digital assays to quantitative analysis ofRNA over wide dynamic range via digital RT-PCR on two rotational devices(Table 1). The first device has a dynamic range (at 95% CI) of 5.2×10²to 4.0×10⁶ molecules/mL with 3-fold resolution and lower detection limitof 1.2×10² molecules/mL. The device was characterized using syntheticcontrol RNA, demonstrating that MV digital RT-PCR performs in agreementwith theoretical predictions over the entire dynamic range (FIG. 3).Results from wells of different volumes were mutually consistent andenabled quantification over a wide dynamic range using only 640 totalwells (FIG. 4). This chip was also validated with viral RNA from two HIVpatients (FIG. 6), demonstrating good agreement with single-pointmeasurements performed on a Roche CAP/CTM v2.0 clinical instrument.Using this chip, positive wells were detected that corresponded to aconcentration of 81 molecules/mL HIV viral RNA purified from patientplasma in the RT-PCR mix, which corresponds to around 37 molecules/mL inthe original plasma samples. While below the detection limit at 95%confidence interval, this concentration should give at least onepositive well 86% of the time, so it is not surprising that all four ofthe experiments had at least one positive well at this concentration.

A second chip was used to test the scalability and flexibility of themultivolume approach by introducing both multiplexed and higher-rangequantification. Additional wells were added with volumes of 0.2 nL and625 nL and divided the device into five individual regions. There was noevidence of cross-contamination among samples on this rotational design,in agreement with previous results on a translational device. Thismultiplexed device was designed to test five samples, each at a dynamicrange (3-fold resolution) from 1.8×10³ to 1.2×10⁷ molecules/mL with alower detection limit of 2.0×10² molecules/mL. Multiplexing capability(FIG. 5) enables a number of features on the same chip, including (i)incorporating negative controls, (ii) measuring levels of control RNA toquantify the quality of sample preparation, (iii) monitoringco-infections, (iv) designing customized arrays for multiple targets,i.e. for nucleic acid targets that require measurements with differentdynamic ranges and resolution, using wells of different sizes withcustomized numbers of wells at each size for each target, and (v)allowing for flexibility depending on technical and economic constraintsby using the same device to perform either more analyses of lowerquality, but at proportionally lower cost, or a single analysis of highquality including wider dynamic range and higher resolution. If thismultiplexed device is used for a single sample, the dynamic range of thedevice with 3-fold resolution is designed to be 1.7×10² to 2.0×10⁷molecules/mL with a lower detection limit of 40 molecules/mL. Even withonly a modest concentrating effect during sample preparation, thisdevice would enable detecting targets at 10-20 molecules/mL in theoriginal sample.

The high sensitivity of the this MV digital RT-PCR platform is valuablefor a number of applications beyond viral load, including detecting rarecells and rare mutations, prenatal diagnostics, and monitoring residualdisease. Besides monitoring the HIV viral load of patients onantiretroviral treatments, this approach is a method to screen newbornswhose mothers are carrying HIV, where maternal HIV antibodies wouldpotentially interfere with the antibody test. In addition, similarmolecular diagnostics methods may be used to measure proviral DNA ininfants. This approach can also be applied to investigation of copynumber variation and gene expression, both for both for research anddiagnostic settings.

The rotational format of the device is useful for resource-limitedsettings because the movement is easy to control even manually; for achip with a 2 in. (50 mm) diameter, a 8° rotation moves the outer edgeof the chip by ˜3.5 mm, a distance that is easily done by hand,especially with internal stoppers and guides. At the same time, thatrotation moves the wells which are 2.8 mm from the center by 0.39 mm.This feature is ideal for multivolume formats but also can be takenadvantage of in single-volume formats. The devices are also particularlyattractive for multivolume formats due to its lack of valves and ease ofoperation. A number of additional developments will increase theusefulness this chip. The considerations among resolution, dynamicrange, and the extent of multiplexing of the multivolume device aredescribed (Kreutz et al., Anal. Chem. 2011, DOI 10.1021/ac201658s). Theexemplary designs presented here were fabricated in glass, and afunctional device of a different design made from plastic by hotembossing was previously demonstrated.

For applications to resource-limited settings, devices made withinexpensive materials such as plastics are suitable. The discloseddevices are compatible with other amplification chemistries, includingpolymerization and depolymerization methods, toe-hold initiatedhybridization-based amplification, and other amplifications includingsilver-based amplification. When combined with isothermal amplificationmethods, such as recombinase polymerase amplification, loop-mediatedamplification, strand-displacement amplification, helicase-dependentamplification, rolling circle amplification, and visual readout methods,the MV digital RT-PCR device makes quantitative molecular diagnosticsaccessible in resource-limited settings.

Chemicals and Materials

All solvents and salts obtained from commercial sources were used asreceived unless otherwise stated. SsoFast EvaGreen SuperMix (2×) waspurchased from Bio-Rad Laboratories (Hercules, Calif.). One-StepSuperScript® III Reverse Transcriptase, iPrep™ purification instrument,and iPrep™ PureLink™ virus kit were purchased from InvitrogenCorporation (Carlsbad, Calif.). All primers were purchased fromIntegrated DNA Technologies (Coralville, Iowa). Bovine serum albumin (20mg/mL) was ordered from Roche Diagnostics (Indianapolis, Ind.). Mineraloil, tetradecane, and DEPC-treated nuclease-free water were purchasedfrom Fisher Scientific (Hanover Park, Ill.). Dichlorodimethylsilane wasordered from Sigma-Aldrich (St. Louis, Mo.). PCR Mastercycler and insitu adapter were purchased from Eppendorf (Hamburg, Germany). Spectrumfood color was purchased from August Thomsen Corp (Glen Cove, N.Y.).Soda-lime glass plates coated with layers of chromium and photoresistwere ordered from Telic Company (Valencia, Calif.). Photomasks weredesigned using AutoCAD (San Rafael, Calif.) and ordered from CAD/ArtServices, Inc. (Bandon, Oreg.). Microposit™ MF™-CD-26 developer waspurchased from Rohm and Hass Electronic Materials LLC (Marlborough,Mass.). Amorphous diamond coated drill bits were purchased from HarveyTool (0.030 inch cutter diameter, Rowley, Mass.). Adhesive PDMS film(0.063 inch thick) was purchased from McMaster (Atlanta, Ga.). TheMinElute PCR purification kit and QIAamp Viral RNA mini kit werepurchased from Qiagen Inc. (Valencia, Calif.). The OptiQuant®-S HCV RNAquantification panel was purchased from AcroMetrix (Benicia, Calif.).

Fabrication of Devices for Multivolume Digital RT-PCR

The procedure for fabricating the devices from soda lime glass was basedon procedures described in previous work. To fabricate devices formultivolume digital RT-PCR, wells of two different depths were etchedusing a two-step exposing-etching protocol. The soda lime glass platepre-coated with chromium and photoresist was first aligned with aphotomask containing the design for wells of 25 nL and 125 nL for Design1 (Table 1, FIG. 11). For Design 2, this photomask also contained thedesigns of the additional wells of 625 nL. The glass plate was thenexposed to UV light using standard exposure protocols. After exposure,the glass plate was detached from the photomask and immersed indeveloper to immediately remove the photoresist that was exposed to UVlight. The underlying chromium layer that was exposed was removed byapplying a chromium etchant (a solution of 0.6:0.365 mol/LHClO4/(NH₄)₂Ce(NO₃)₆). The glass plate was thoroughly rinsed with waterand dried with nitrogen gas. The glass plate was then aligned with asecond photomask containing the designs of wells of 1 nL and 5 nL forDesign 1 (Table 1, FIG. 11) by using a mask aligner. For Design 2 (FIG.11), this second photomask also contained the designs of the additionalwells of 0.2 nL. The glass plate was then exposed to UV light a secondtime. After the second exposure, the photomask was detached from theglass plate, and the back side of the glass plate was protected with PVCsealing tape. The taped glass plate was then immersed in a glass etchingsolution (1:0.5:0.75 mol/L HF/NH₄F/HNO₃) to etch the glass surface wherechromium coating was removed in the previous step (areas containingwells of 25 nL, 125 nL, and 625 nL), and the etching depth was measuredby a profilometer. After the larger features were etched to a depth of70 μm, the glass plate was placed in the developer again to remove thepreviously exposed photoresist in areas containing the patterns for thesmaller features (1 nL and 5 nL wells, and the additional wells of 0.2nL for Design 2, FIG. 11). The underlying chromium layer was removed byusing the chromium etchant as describe above, and a second glass etchingstep was performed to etch all features to a further depth of 30 μm. Thefinal device contained wells of depths of 100 μm and 30 μm wasfabricated.

After the two-step etching, the glass plate was thoroughly rinsed withMillipore water and ethanol and then dried with nitrogen gas. The glassplate was oxidized using a plasma cleaner and immediately placed in adesicator with dichlorodimethylsilane for gas-phase silanization. ForDesign 2A (FIG. 11), circular inlet reservoirs (4 mm inner diameter and6 mm outer diameter) were made by cutting adhesive PDMS film, thenfixing the reservoirs around the five inlets before plasma cleaning.After one hour, the silanized glass plate was thoroughly rinsed withchloroform, acetone, and ethanol, and then dried with nitrogen gas.

To re-use the glass devices, each device was thoroughly cleaned withpiranha acid (3:1 sulfuric acid: hydrogen peroxide), then oxidized usinga plasma cleaner and silanized as described above.

Device Assembly

Devices were assembled under de-gassed oil (mineral oil: tetradecane 1:4v/v). The bottom plate was immersed into the oil phase with thepatterned wells facing up, and the top plate was then immersed into theoil phase and placed on top of the bottom plate with the patterned sidefacing down. The two plates were aligned under a stereoscope (Leica,Germany) as shown in FIG. 1A and stabilized using binder clips.

Device Loading

A through-hole was drilled in the center of the top plate to serve asthe solution inlet for Design 1 and Design 2B. The reagent solution wasloaded through the inlet by pipetting. For Design 2A, five through-holeswere drilled at the top left corner of the top plate to serve as fluidinlets (FIG. 5A). For multiplex experiments, five different reactionsolutions were placed in the inlet reservoirs, and a dead-end fillingadapter was placed on top of the devices to cover all the inlets. Apressure of 18 mmHg was applied to load all the solutionssimultaneously. The principle and detailed method for dead-end fillingare described in a previous work.³ Reservoirs were removed after thesolution was loaded.

Synthesis and Purification of Control RNA (906nt)

The control RNA (906 nucleotide) was synthesized from the LITMUS 28iMalControl Plasmid using a HiScribe™ T7 In Vitro Transcription Kit with themanufacture's recommended procedures (New England Biolabs, Ipswich,Mass.) and purified using MinElute PCR purification kit with manufacturerecommended protocols.

Automatic Viral RNA Purification from Plasma Sample

Plasma samples containing the HIV virus were obtained from deidentifiedpatients at the University of Chicago Hospital. Plasma containing amodified HCV virus as a control (25 million IU/mL, part of OptiQuant-SHCV Quantification Panel) was purchased from AcroMetrix (Benicia,Calif.). A plasma sample of 400 μL was mixed with 400 μL lysis buffer(Invitrogen Corporation, Carlsbad, Calif.) to lyse the virus. Then 2 μLof control RNA (906 nt) was added to characterize the purificationefficiency and concentrating factor. The mixed sample was thentransferred into the iPrep™ PureLink™ virus cartridge. The cartridge wasplaced in the iPrep™ purification instrument and the purificationprotocol was performed according to the manufacturer's instructions. Thefinal elution volume was 50 μL, therefore a theoretical eight-foldconcentrating factor was expected. The initial concentration of controlRNA and the concentration of control RNA in the purified sample afterpreparation were characterized on the device (Design 1). The finalconcentrating factor was 4.5 for HCV and 6.6 for HIV in the multiplexRT-PCR amplification (FIG. 5). The concentrating factors for the two HIVsamples were 7.1 and 6.6 for the experiments in FIG. 6.

Primer Sequences for RT-PCR Amplification

Primers for the control RNA (906 nt) were: GAA GAG TTG GCG AAA GAT CCACG (SEQ ID NO: 1) and CGA GCT CGA ATT AGT CTG CGC (SEQ ID NO: 2). Thecontrol RNA template was serially diluted in 1 mg/mL BSA solution. TheRT-PCR mix contained the following: 30 μL of 2× EvaGreen SuperMix, 1 μLof each primer (10 μmol/L), 3 μL of BSA solution (20 mg/mL), 1.5 μL ofSuperScript® III Reverse Transcriptase, 17.5 μL of nuclease-free water,and 6 μL of template solution.

Primer sequences for HIV viral RNA was selected from a previouspublication:⁴ GRA ACC CAC TGC TTA ASS CTC AA (SEQ ID NO: 3); GAG GGA TCTCTA GNY ACC AGA GT (SEQ ID NO: 4). Primer sequences for control HCVviral RNA were selected from a previous publication:⁵ GAG TAG TGT TGGGTC GCG AA (SEQ ID NO: 5); GTG CAC GGT CTA CGA GAC CTC (SEQ ID NO: 6).

RT-PCR Amplification on the Devices

To amplify HIV viral RNA in FIG. 5, the RT-PCR mix contained thefollowing: 15 μL of 2× EvaGreen SuperMix, 0.6 μL of each primer (10μmol/L), 1.5 μL of BSA solution (20 mg/mL), 0.75 μL of SuperScript® IIIReverse Transcriptase, 10.05 μL of nuclease-free water, and 1.5 μL oftemplate solution. The template solution used here was diluted 250-foldfrom the original HIV viral RNA stock solution purified from Patientsample 2 using 1 mg/mL BSA solution.

To amplify control HCV viral RNA in FIG. 5, the RT-PCR mix contained thefollowing: 15 μL of 2× EvaGreen SuperMix, 0.25 μL of each primer (10μmol/L), 1.5 μL, of BSA solution (20 mg/mL), 0.75 μL of SuperScript® IIIReverse Transcriptase, 10.25 μL of nuclease-free water, and 2 μL oftemplate solution. The template solution was diluted 5-fold from theoriginal control HCV viral RNA stock solution purified from OptiQuant-SHCV Quantification Panel.

To amplify the control RNA (906 nt) in FIG. 5, the RT-PCR mix containedthe following: 15 μL of 2× EvaGreen SuperMix, 0.25 μL of each primer (10μmol/L), 1.5 μL of BSA solution (20 mg/mL), 0.75 μL of SuperScript® IIIReverse Transcriptase, 10.25 μL of nuclease-free water, and 2 μL oftemplate solution. The template solution was diluted 5-fold from theoriginal control HCV viral RNA stock solution purified from OptiQuant-SHCV Quantification Panel.

The experiment in FIG. 5 was repeated six times, and the resultant datawere used to calculate the target concentration.

To amplify HIV viral RNA with expected final concentration above 1000molecules/mL in the RT-PCR mix in FIG. 6, the RT-PCR mix contained thefollowing: 20 μL of 2× EvaGreen SuperMix, 1 μL of each primer (10μmol/L), 2 μL of BSA solution (20 mg/mL), 1 μL of SuperScript® IIIReverse Transcriptase, 13 μL, of nuclease-free water, and 2 μL oftemplate solution. The template was serially diluted in 1 mg/mL BSAsolution. For experiments with HIV viral RNA concentration below 1000molecules/mL in the final RT-PCR mix, the RT-PCR mix contained thefollowing: 30 μL of 2× EvaGreen SuperMix, 1.5 μL of each primer (10μmol/L), 2 μL of BSA solution (20 mg/mL), 1.5 μL of SuperScript® IIIReverse Transcriptase, 3.5 μL of nuclease-free water, and 20 μL oftemplate solution.

To amplify the control RNA (906 nt) in the HIV sample in FIG. 5 and FIG.6, the RT-PCR mix contained the following: 20 μL of 2× EvaGreenSuperMix, 1 μL of each primer (10 μmol/L), 2 μL of BSA solution (20mg/mL), 1 μL of SuperScript® III Reverse Transcriptase, 13 μL ofnuclease-free water, and 2 μL of HIV viral RNA stock solution aftersample preparation.

The concentration of control RNA (906 nt) before sample preparation wascharacterized on device Design 1 (FIG. 11) with the RT-PCR mix containedthe following: 20 μL of 2× EvaGreen SuperMix, 1 μL of each primer (10μmol/L), 2 μL of BSA solution (20 mg/mL), 1 μL of SuperScript® IIIReverse Transcriptase, 13 μL of nuclease-free water, and 2 μL oftemplate solution. The template was prepared by diluting 2 μL of stockcontrol RNA (906nt) solution into 400 μL of 1 mg/mL BSA solution.

To amplify HIV viral RNA in FIG. 7, the RT-PCR mix for HIV viral RNAcontained the following: 90 μL of 2× EvaGreen SuperMix, 3.6 μL of eachprimer (10 μmol/L), 6 μL of BSA solution (20 mg/mL), 4.5 μL ofSuperScript® III Reverse Transcriptase, 12.3 μL of nuclease-free water,and 60 μL of template solution. The template solution used here wasdiluted 62500-fold from the original HIV viral RNA stock solutionpurified from Patient sample 2 using 1 mg/mL BSA solution. Thisexperiment was repeated six times and all data was used to calculate HIVviral RNA concentration. Three negative control experiments wereperformed with the same primer pairs but no HIV viral RNA, and showed nofalse positives.

The amplifications were performed using a PCR mastercycler machine(Eppendorf). To amplify the RNA, an initial 30 min at 50° C. was appliedfor reverse transcription, then 2 min at 95° C. for enzyme activation,followed by 35 cycles of 1 min at 95° C., 30 sec at 55° C. and 45 sec at72° C. After the final cycle, a final elongation step was applied for 5min at 72° C. This thermal cycling program was applied to allexperiments except for those in FIG. 7, where 39 cycles were adaptedinstead of 35 cycles.

Image Acquisition and Analysis

Bright-field images in FIG. 1 and FIG. 5 were acquired using a Canon EOSRebel XS digital SLR camera (Lake Success, N.Y.). Other bright-fieldimages were acquired using a Leica stereoscope. All fluorescence imageswere acquired by Leica DMI 6000 B epi-fluorescence microscope with a5×/0.15 NA objective and L5 filter at room temperature. All fluorescenceimages were corrected for background by using an image acquired with astandard fluorescent control slide. All the images were then stitchedtogether using MetaMorph software (Molecular Devices, Sunnyvale,Calif.).

FIG. 7 shows a representative experiment performing RT-PCR of HIV viralRNA at an expected concentration of 51 molecules/mL in RT-PCR mix on theDesign 2B device to test the lower detection limit of the device. Thisexperiment was repeated six times to quantify the viral RNAconcentration.

FIG. 8 shows a representative negative control for HIV viral load (HIVprimers with no loaded HIV RNA template) on device Design 1,corresponding to experiments shown in FIG. 6.

FIG. 9 (table) presents performance of quantification of HIV viral RNAconcentration from patient 1 on device comparing to Roche COBAS®AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0 system (CAP/CTM v2.0). Eachexperiment was repeated at least four times on device. Only 2significant digits are shown. The expected HIV concentration of patientplasma was calculated based on dilution factors and a single result fromRoche CAP/CTM v2.0. The results from device are obtained with serialdiluted purified patient HIV viral RNA and are converted to the originalconcentration in patient plasma (with or without dilutions) using thepurification concentrating factor.

FIG. 10 (table) shows performance of quantification of HIV viral RNAconcentration from patient 2 on device comparing to Roche COBAS®AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0 system (CAP/CTM v2.0). Eachexperiment was repeated at least four times on device. Only 2significant digits are shown. The expected HIV concentration of patientplasma was calculated based on dilution factors and a single result fromRoche CAP/CTM v2.0. The results from device are obtained with serialdiluted purified patient HIV viral RNA and are converted to the originalconcentration in patient plasma (with or without dilutions) using thepurification concentrating factor.

LAMP Amplification

Digital reverse transcription loop mediated isothermal amplification(RT-LAMP) can be performed on a device according to the presentdisclosure. In some embodiments, digital RT-LAMP is performed on amultivolume device. In one embodiment, one-step digital RT-LAMP iscarried out by mixing template, primers, detection reagent, reaction mixand enzyme, then loading the solution onto a device and heating up thedevice to a proper temperature for a period of time.

For example, the following mixture of reagents has been used: 20 μLreaction mix, 2 μL enzyme mix (Loopamp RNA Amplification Kit from EikenChemical Co., LTD.), 2 μL detection reagent (Eiken Chemical Co., LTD.),2 μL, 20 mg/mL BSA, 8 μL RNase free water, 4 uL primer mix and 2 μL HIVRNA purified from AcroMetrix® HIV-1 Panel 1E6. The final concentrationof primers was 2 μM for BIP/FIP, 1 μM for LOOP primers, 0.25 μM forB3/F3. All solutions were operated on ice.

The solution was loaded onto a multivolume device (design published inShen et al., JACS 2011 133: 17705) and the relative position of theplates of the device were fixed by wax. The whole device was heated on athermal cycler block (Eppendorf) for about 1 hour then terminated at 95°C. for 2 minutes. The fluorescence image was acquired by Leica DMI 6000B epi-fluorescence microscope with a 5×/0.15 NA objective and L5 filterat room temperature. The measured concentration of digital RT-LAMP was10% of that from digital RT-PCR using B3/F3 as primers.

In another embodiment, two-step digital RT-LAMP is carried out in twoseparate steps. Reverse Transcription is done by mixing template,BIP/FIP primers, reverse transcriptase, and reaction mix in a tube, andheating to a proper temperature. Digital LAMP is performed by mixingcDNA solution with all other components, loading the solution onto adevice, and heating the device at a proper temperature for a period oftime.

In another embodiment, digital RT-LAMP is performed by running thereverse transcription step on the device in a digital format, mixing theproduct with other components of LAMP on-chip and heating the device.The result of this protocol has been experimentally observed to be thesame as when performing the RT step in a test tube.

In one set of experiments performed with two-step digital RT-LAMP, 10 μLreaction mix, 1 μL 20 mg/mL BSA, 0.5 μL Superscript III reversetranscriptase (Invitrogen), 6 μL RNase free water, 0.5 uL BIP/FIP primermix (10 μM) and 2 μL HIV RNA purified from AcroMetrix® HIV-1 Panel 1E6were mixed together in a test tube. All solutions were operated on ice.The solution was heated to 50° C. for 15 min for reverse transcription.

All other components of LAMP mixture (2 μL enzyme mix, 2 μL detectionreagent, 10 μL reaction mix, 1 μL 20 mg/mL BSA, all other primers andRNase free water to make up the volume to 20 μL.) were mixed togetherwith the solution obtained from reverse transcription and loaded on adevice immediately. The whole device was heated on a thermal cyclerblock (Eppendorf) for about 1 hour then terminated at 95° C. for 2minutes. Imaging settings were the same as described for the one-stepRT-LAMP experimental protocol above. The measured concentration obtainedafter performing digital RT-LAMP was found to be 30% of that fromdigital RT-PCR using B3/F3 as primers.

In another set of experiments, the efficiency of two-step digitalRT-LAMP was found to be improved by adding only BIP/FIP primer in the RTstep, adding RNase H after the RT step and removing B3 from the primermixture.

For example, 10 μL reaction mix, 1 μL 20 mg/mL BSA, 0.5 μL SuperscriptIII reverse transcriptase (Invitrogen), 6 μL RNase free water, 0.5 uLBIP/FIP primer mix (10 μM) and 2 μL HIV RNA purified from AcroMetrix®HIV-1 Panel 1E6 were mixed together. All solutions were operated on ice.The solution was heated to 50° C. for 15 min for reverse transcriptionthen followed by the addition of 0.5 μL RNase H (NEB) and incubation at37° C. for 10 minutes.

All other components of LAMP mixture (2 μL enzyme mix, 2 μL detectionreagent, 10 μL reaction mix, 1 μL 20 mg/mL BSA, all other primers exceptfor B3 and RNase free water to make up the volume to 20 μL) were mixedtogether with the solution obtained from reverse transcription andloaded on a device immediately. Heating and imaging settings were thesame as described for the two-step RT-LAMP experimental protocol above.The measured concentration after performing digital RT-LAMP was found tobe 60% of that obtained via digital RT-PCR using B3/F3 as primers.

In another set of experiments, the efficiency of two-step digitalRT-LAMP was found to be improved by adding only BIP/FIP primer in the RTstep, adding thermostable RNase H into the LAMP mixture and removing B3from the primer mixture.

For example, 10 μL reaction mix, 1 μL 20 mg/mL BSA, 0.5 μL SuperscriptIII reverse transcriptase (Invitrogen), 6 μL, RNase free water, 0.5 uLBIP/FIP primer mix (10 μM) and 24 HIV RNA purified from AcroMetrix®HIV-1 Panel 1E6 were mixed together. All solutions were operated on ice.The solution was heated to 50° C. for 15 min for reverse transcription.

All other components of LAMP mixture (2 μL enzyme mix, 2 μL detectionreagent, 10 μL reaction mix, 1 μL 20 mg/mL BSA, all other primers exceptfor B3 and RNase free water to make up the volume to 20 μL) and 0.5 uLHybridase™ Thermostable RNase H (Epicenter) were mixed together with thesolution obtained from reverse transcription and loaded on a deviceimmediately. The heating and imaging settings were the same as describedfor the two-step RT-LAMP experimental protocols above. The measuredconcentration after performing digital RT-LAMP was found to be 60% ofthat obtained from digital RT-PCR using B3/F3 as primers.

Imaging with Mobile Device Camera

In one embodiment, an imaging device with wireless communicationcapability may be used to capture the results of both isothermal andnon-isothermal methods such as digital LAMP and digital NASBA performedon a microfluidic device as disclosed herein.

As one example, an iPhone 4S™ is used to capture results on a discloseddevice. The fluorescence readout is achieved by a standard iPhone 4S™8MP camera equipped with a yellow dichroic long-pass filter 10CGA-530(Newport, Franklin, Mass.). Fluorescence excitation was achieved byshining blue light on a device at an oblique angle of approximately 30°.The light source was a blue LED (LIU003) equipped with a blue short-passdichroic filter FD1B (Thorlabs, Newton, N.J.). Excitation light reachedthe sample in two ways: by direct illumination and by multiplereflections between the device plates.

A device of a design described in a previous publication (Shen et al.,JACS 2011 133: 17705) was imaged in the experiments. Soda-lime glassplates with chromium and photoresist coating (Telic Company, Valencia,Calif.) were used to fabricate devices. The method for making a glassdevice described in a previous publication (Du, Lab Chip 2009,2286-2292), was used. Briefly, the photoresist-coated glass plate wasexposed to ultraviolet light covered by a photomask with designs of thewells and ducts. Following removal of the photoresist using 0.1 M NaOHsolution, the exposed chromium coating was removed by a chromium-etchingsolution. The patterns were then etched in glass etching solution in a40° C. shaker. After glass etching, the remaining photoresist andchromium coatings were removed by ethanol and chromium-etching solution,respectively. The surfaces of the etched glass plates were cleaned andsubjected to an oxygen plasma treatment, and then the surfaces wererendered hydrophobic by silanization in a vacuum desiccator aspreviously described (Roach, Analytical Chemistry 2005, 785-796). Inletholes were drilled with a diamond drill bit 0.035 inch in diameter.

A fluorescent reaction mix for digital LAMP was prepared, loaded in thedevice, and allowed to react, as described elsewhere in thisapplication.

An image was produced using an iPhone application, Camera+™ (obtainedvia taptaptap.com) in automatic mode; no tripod was used. The excitationlight was shined from one side of the device under an oblique angle ofapproximately 30°. The resulting illumination was relatively uniform,suggesting that light spreads by multiple reflections inside theanalysis device.

FIG. 12 shows an image of a multivolume device filled with LAMP reactionmix obtained with a iPhone 4S™ camera. Image size is 8 MP. The totalnumber of wells of each kind is 160. In total there are 122 positivelargest wells, 42 of the second largest positive wells, 5 of the secondsmallest positive wells and 2 of the smallest positive wells. Well countwas done automatically using Metamorph software. The signal/noise ratiois over 20 even for the smallest wells.

FIG. 13 shows a magnified portion of the image in FIG. 12. In thisimage, the smallest wells in the image are approximately 15-20 pixelswide and the signal/noise ratio is over 20.

Additional information may be found in the following references, each ofwhich is incorporated by reference in its entirety.

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What is claimed is:
 1. A method, comprising: a) introducing a samplecomprising a target molecule into a device comprising a first componentcomprising a plurality of first areas, a second component comprising aplurality of second areas, wherein the first and second components areconfigured to slip relative to the other between a first position,wherein the plurality of first areas and the plurality of second areasoverlap to form a continuous fluidic path, and a second position,wherein at least two areas of said plurality of first areas and secondareas are isolated from each other; b) in said first position,distributing an amount of said target molecule into said at least twoareas of the plurality of first areas and second areas via thecontinuous fluidic path extending through the overlapping plurality offirst areas and second areas and which connects the at least two areas,wherein said at least two areas define volumes that differ from oneanother; c) slipping said first or second component relative to theother to the second position, thereby isolating the at least two areas;(d) effecting a reaction on said amount of said target molecule in saidat least two isolated areas and thereby producing a reaction product insaid at least two isolated areas; (e) detecting said reaction productoptically in said at least two areas; and (f) estimating, from saidreaction product, a level of said target molecule in said sample.
 2. Themethod of claim 1, wherein said target molecule comprises a nucleicacid.
 3. The method of claim 2, wherein effecting the reaction comprisescontacting an amplification reagent with said nucleic acid.
 4. Themethod of claim 2, wherein at least one of said at least two areas isestimated to comprise about one molecule of nucleic acid.
 5. The methodof claim 1, wherein at least one of said at least two areas is estimatedto contain only target molecule.
 6. The method of claim 1, wherein saidreaction comprises nucleic acid amplification.
 7. The method of claim 6,wherein said nucleic acid amplification comprises polymerase chainreaction, room-temperature polymerase chain reaction, nested polymerasechain reaction, multiplex polymerase chain reaction, arbitrarily primedpolymerase chain reaction, nucleic acid sequence-based amplification,transcription mediated amplification, strand displacement amplification,branched DNA probe target amplification, ligase chain reaction, cleavaseinvader amplification, anti DNA-RNA hybrid antibody amplification, orany combination thereof.
 8. The method of claim 6, wherein said nucleicacid amplification is essentially isothermal.
 9. The method of claim 1,wherein at least one of said at least two areas defines a volume in therange of from about 1 picoliter to about 1 microliter.
 10. The method ofclaim 1, wherein distribution comprises effecting relative motionbetween the first and second component so as to distribute said amountof said target molecule into said at least two areas.
 11. The method ofclaim 10, wherein said relative motion gives rise to said amount of saidtarget molecule being divided among at least 10 areas.
 12. The method ofclaim 11, wherein said relative motion gives rise to said amount of saidtarget molecule being divided among at least 50 areas.
 13. The method ofclaim 1, wherein said reaction is effected at two or more areasessentially simultaneously.
 14. The method of claim 1, wherein effectingthe reaction comprises heating said amount of said target molecule. 15.The method of claim 1, wherein the introducing the sample comprising atarget molecule into the device comprises introducing into an inlet inat least one of the first or second components of the device the samplecomprising the target molecule.
 16. The method of claim 1, whereineffecting the reaction comprises contacting an amplification reagentwith said amount of said target molecule in said at least two areas byeffecting relative motion between the first and second components of thedevice.